Salvador tumor suppressor gene

ABSTRACT

The invention relates to the discovery of a novel  Drosophila  gene, salavador, and to the discovery of a tumor suppressor function for its human counterpart. The salvador nucleic acid and protein molecules, their use in the diagnosis and treatment of disorders characterized by aberrant salvador molecule expression, as well as various research uses are described.

RELATED APPLICATIONS

This application claims priority to U.S. Application Ser. No.60/348,432, filed Oct. 26, 2001, which is hereby incorporated byreference.

GOVERNMENT SUPPORT

This invention was made in part with support from the NationalInstitutes of Health, a United States Government Agency, under grantnumbers EY11632, GM61672, and CA87691. The Government may have certainrights in this invention.

FILED OF THE INVENTION

This invention relates to the discovery of a novel Drosophila gene,Salvador, and to the discovery of a tumor suppressor function for itshuman counterpart. The invention is directed to the isolated tumorsuppressor nucleic acids, the proteins encoded by these nucleic acids,binding agents that selectively bind thereto, and various diagnostic,therapeutic and research uses of these compositions.

BACKGROUND OF THE INVENTION

The number of cells in an organism is determined by the number of cellsgenerated as a result of cell proliferation as well the number of cellsthat are eliminated by cell death. Both cell proliferation and celldeath are strictly regulated by developmental mechanisms to ensure thatan organ of a characteristic shape and size is generated. The verymechanisms that regulate normal growth and cell proliferation are oftenthose that are perturbed in human cancers. Mutational events found incancers can either promote growth and cell proiferation or impede celldeath.

Cancer progression is caused by accumulation of multiple mutations thatprovide selective advantage during cancer growth, invasion andmetastasis (Haber, D. A. and Fearon, E. R. Lancet 351 Suppl 2:SII1-8,1998; Loeb, K. R. and Loeb, L. A., Carcinogenesis 21(3):379-85, 2000;Loeb, L. A., et al. Cancer Research 34 (9):2311-21, 1974). While gain offunction mutations occur in oncogenes, many of the genetic events thatunderlie cancer appear to be inactivating, or loss of functionmutations, affecting tumor suppressor genes (Haber, D. A. and Fearon, E.R. Lancet 351 Suppl 2:SII1-8, 1998). Tumor suppressor genes identifiedto date exhibit diverse cellular functions (Haber D. and Harlow E.Nature Genetics 16 (4):320-2, 1997).

Functional studies on these tumor suppressor genes have supported theoriginal hypothesis that these genes represent potential bottlenecks inwide variety of cellular pathways, including proliferation,differentiation, apoptosis and response to DNA damage (Haber D. andHarlow E. Nature Genetics 16 (4):320-2, 1997). For example, p53 and WT1are DNA binding transcription factors; RB1, APC and possibly BRCA1indirectly modulate transcription; P16 is an inhibitor of kinasesrequired for cell cycle progression; PTEN is a novel phosphatase; NF2 isa cell structural component; and VHL is a potential mediator of mRNAprocessing. A large number of genes are believed to be genomiccaretakers and mutations in these genes cause microsatellite instability(e.g., MSH2, MLH1, PMS1 and PMS2) or chromosomal instability (e.g., p53,possibly BRCA1 and BRCA2). To date, genes involved in advanced stages ofcancer progression such as invasion, angiogenesis and metastasis havenot been identified (Haber D. and Harlow E. Nature Genetics 16(4):320-2, 1997; Fearon, E. R. Current Biology 9 (23):R873-5, 1999).They are likely to become evident over time with large-scale genome wideanalysis.

The vast majority of cancers result from sporadic genetic events andonly rare cases (fewer than 1%) have an inherited component (Fearon, E.R. Science 278 :1043-50, 1997; Kinzler, K. W. and Vogelstein B. Cell. 87(2):159-70, 1996). However, the isolation of tumor suppressor genes hastypically originated from genetic analysis of such rare inherited cancersyndromes (Fearon, E. R. Science 278 :1043-50, 1997). Linkage analysison large families with cancer present in multiple generations allowsidentification of markers that co-segregate with cancer. In some casescytogenetic abnormalities could also be observed either in sporadic orin germline tumors (Fearon, E. R. Science 278 :1043-50, 1997; Gray, J.W. and Collins, C. Carcinogenesis. 21 (3):443-52, 2000). For example, asmall fraction of retinoblastomas have a homozygous deletion of RB 1gene (Fearon, E. R. Science 278 :1043-50, 1997). Rare Wilms tumor andcolon cancer have deletions of WT1 and APC, respectively. These germlineor sporadic homozygous deletions have been instrumental in tumorsuppressor gene cloning efforts (Fearon, E. R. Science 278 :1043-50,1997). Allelic losses in tumors are typically detected as “loss ofheterozygosity” or “LOH.” This represents loss of a polymorphic marker,commonly resulting from a large interstitial deletion or chromosomalnon-disjunction event. While LOH is a common event in cancer, it onlyallows rough mapping of tumor suppressor loci (Gray, J. W. and Collins,C. Carcinogenesis. 21 (3):443-52, 2000). The large size of the LOHregion (>10 Mb) makes the identification of the specific tumorsuppressor gene targeted by mutation difficult. In contrast, homozygousdeletions in tumors are typically small (<100 Kb) since they arerestricted by the deletion of the flanking genes. Homozygous deletionsoccur by diverse mechanisms, including a small deletion in one alleleaccompanied by LOH of the second allele, or even large deletion of eachallele whose common region of overlap is small. Identification of suchhomozygous deletions can be a powerful approach to identify tumorsuppressor genes (Fearon, E. R. Science 278:1043-50, 1997).

Significant technological advances have been made to identify regions ofchromosomes involved in tumor progression. Analyses of metaphasechromosomes show chromosomal rearrangements in leukemia and lymphomas(Rowley, J. D. Annu. Rev. Genet. 32 :495-519). This is more difficult insolid tumors where karyotyping is less commonly performed. Fluorescencein situ hybridization (FISH) has greatly improved the sensitivity andspecificity of detecting chromosome aberrations (Pinkel, D., et al.Proc. Natl. Acad. Sci. 85 9138-9142, 1988; Speicher, M. R. and Ward D.C. Nature Med. 2:1046-1048 1996). However, its application in humanmalignancies is still limited because of the complex karyotypes seen inclinical samples. Comparative genome hybridization (CGH) uses bothnormal and tumor genomes to identify regions in tumor DNA that haveundergone changes in copy number (Kallioniemi, O. P., et al. Seminars inCancer Biology. 4 (1):41-6, 1993). In this technique, normal and tumorDNA are labeled with two different haptens that fluoresce at differentwavelengths. The probes are then hybridized to metaphase chromosomes inthe presence of excess Cot-i DNA thus inhibiting hybridization oflabeled repetitive sequences. The ratio of the amount of two genomesthat hybridize to specific areas of the chromosomes indicates the copynumber of the two samples. CGH is currently limited to a resolution of10 to 20 Mb and more sensitive in detecting amplifications rather than asmall deletion (Gray, J. W. and Collins, C. Carcinogenesis. 21(3):443-52, 2000). An alternative method, Representational DifferenceAnalysis (RDA) is a PCR based subtractive hybridization technique, thatis particularly applicable in isolating homozygous deletions in tumors(Lisitsyn, N., et al. Science. 259 (5097):946-51, 1993; Lisitsyn N A.Trends in Genetics 11 (8):303-7, 1995; Lisitsyn, N. and Wigler, M.Methods in Enzymology. 254:291-304, 1995). It has already beensuccessful in isolating tumor suppressor genes PTEN and DMBT1 and hasplayed a significant role in cloning of BRCA2 (Mollenhauer, J., et al.Nature Genetics. 17 (1):32-9, 1997; Li, J., et al. Science 275,1943-1947, 1997; Schutte, M., et al. Proc. Natl. Acad Sci. 92,5950-5954, 1995).

The Drosophila compound eye is particularly suited to the application ofgenetic approaches to the study of cell proliferation and cell death inthe context of organ development (Wolff, T. and Ready, D. F. (1993)Pattern formation in the Drosophila retina. In The Development ofDrosophila melanogaster, M. Bate, and A. Martinez Arias, eds.(Plainview, N.Y.: Cold Spring Harbor Laboratory Press) 1277-1325). Theadult eye develops from a primordium consisting of approximately 30cells in the embryo. Cell growth and proliferation occur during allstages of larval development. Most of the cells generated lo adoptspecialized fates (e.g., photoreceptor, pigment cell) during the latelarval and pupal stages, leaving approximately 2000 unspecified cells.These excess cells are subsequently eliminated by a wave of apoptosis.Thus, the final number of cells in the adult eye can be altered bychanges in either cell proliferation or cell death.

While the developmental signals that trigger cell cycle exit orapoptosis in Drosophila are still poorly characterized, considerableprogress has been made in identifying the endpoints of these pathways.In many different tissues, cell cycle exit appears to be contingent onthe downregulation of Cyclin E levels (Knoblich, J. A., et al. (1994)Cell 77, 107-120). This coincides with increased expression of the cdkinhibitor Dacapo during the final cell cycle (de Nooij, J. C., et al.(1996) Cell 87, 1237-1247; Lane, M. E., et al. (1996) Cell 87,1225-1235). Dacapo inactivates residual Cyclin E/cdk2 complexes andfacilitates a precisely timed exit from the cell cycle. The decrease inCyclin E Summary levels is primarily achieved by a reduction in itstranscription, but other mechanisms including degradation of Cyclin Eprotein appear to be important (Jones, L., et al. (2000) Development127, 46194630; Moberg, K. H., et al. (2001) Nature 413, 311-316).Developmentally regulated cell death in the pupal retina is mediated bycaspase activation. The Reaper, Hid, and Grim proteins bind to theDrosophila inhibitor of apoptosis 1 (DIAP1) protein and prevent DIAP1from inhibiting caspases (Goyal, L., et al. (2000) EMBO J 19, 589-597;Lisi, S., et al. (2000) Genetics 154, 669-678; Wang, S. L. (1999) Cell98, 453-463).

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery of a novel Drosophilagene, termed salvador, which is mutated in tumorous outgrowths (GenbankAccession Number AY131211). Three mutations were identified in thesalvador locus that give mutant Drosophila cells a proliferativeadvantage with respect to wild type cells. The Drosophila salvador genewas cloned and found to encode a novel protein having two WW domainswith highly conserved orthologues in C. elegans and mammals. The threesalvador mutations identified in Drosophila result in proteins lackingboth WW domains.

The human homologue of salvador, hWW45 (GenBank accession numberNM0218118), has been identified, but its function was previously unknown(Valverde, P. Biochem. Biophys. Res. Commun. 276: 990-998, 2000). TheDrosophila and human salvador gene products share 47% identity and 54%similarity over the C-terminal 188 amino acids. The invention also isbased, in part, on the discovery that the human counterpart of theDrosophila gene is deleted or mutated in at least three tumor-derivedcancer cell lines. Accordingly, although not wishing to be bound to anyparticular theory or mechanism, it is believed that the salvador genesfrom various species play a role in modulating cellular development(including cell proliferation, growth, and death) by regulating bothcell cycle exit and apoptosis. Thus, the invention is directed to novelcompositions of Salvador human and Drosophila nucleic acids and proteinsencoded thereby (SEQ ID NOs: 1, 2, 3, and 4), as well as to agents thatselectively bind to these novel molecules. Further, the inventionincludes diagnostic, therapeutic, and research applications of thesecompositions.

According to one aspect of the invention, an isolated nucleic acidmolecule is provided. The isolated nucleic acid molecule is selectedfrom:

-   -   (a) nucleic acid molecules which hybridize under stringent        conditions to a nucleic acid molecule having a nucleotide        sequence set forth as SEQ ID NO:1 (human salvador cDNA) or SEQ        ID NO:3 (Drosophila salvador cDNA), and which code for a        Salvador protein,    -   (b) deletions, additions and substitutions of the nucleic acid        molecules of (a), which code for a Salvador protein,    -   (c) nucleic acid molecules that differ from the nucleic acid        molecules of (a) or (b) in codon sequence due to the degeneracy        of the genetic code, or    -   (d) complements of (a), (b) or (c).

The preferred isolated nucleic acids of the invention are salvadornucleic acid molecules which encode a Salvador protein and includenucleic acid molecules that encode both wild-type and mutant Salvadorproteins. As used herein, a Salvador protein refers to a protein whichis encoded by a nucleic acid having SEQ ID NO:1 or SEQ ID NO:3; afunctional fragment or equivalent thereof, provided that the functionalfragment or equivalent encodes a protein which modulates cellulardevelopment by, for example, modulating cell maturation/differentiation,cell growth, cell proliferation, and/or cell death; wild-type Salvadorproteins; and mutant Salvador proteins. Methods to determine whether aprotein modulates cellular development involve introducing the protein(or the nucleic acid which encodes the protein) into the cell andobserving a change in cellular development in accordance with standardprocedures known to those of ordinary skill in the art. Such methods canbe employed in vivo (e.g., observing tumor suppression) as well as invitro (e.g., apoptosis assays). In a preferred embodiment, the isolatednucleic acid molecule is SEQ ID NO:1 or SEQ ID NO:3.

Other preferred isolated nucleic acids are fragments comprising one ormore WW domains of a salvador gene or functional portions thereof.

According to another aspect of the invention, further isolated nucleicacid molecules that are based on the above-noted salvador nucleic acidmolecules are provided. In this aspect, the isolated nucleic acidmolecules are selected from: (a) a unique fragment of the nucleotidesequence set forth as SEQ ID NO:1 or set forth as SEQ ID NO:3 between 12and Is 2000 nucleotides in length (more preferably, between 12 and 1400nucleotides in length and, most preferably, between 12 and 32nucleotides in length) or more and (b) complements of (a), wherein theunique fragments exclude nucleic acids having nucleotide sequences thatare contained within SEQ ID NO:1 or SEQ ID NO:3, and that are known asof the priority date of this application. Known fragments, i.e.,fragments that are not unique, of SEQ ID NO:1 and SEQ ID NO:3 areprovided in Table 1 and identified by their positions in SEQ ID NO:1 andSEQ ID NO:3.

In a preferred embodiment, the unique fragments comprise the sequencesselected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ IDNO:14.

In yet another aspect of the invention, Mutant Salvador nucleic acidmolecules are provided. The Mutant salvador nucleic acid moleculescontain a sequence which is the same as SEQ ID NO:1 or SEQ ID NO:3, withthe exception that the sequence includes one or more mutations, e.g.,point mutations, deletion mutations, or truncations, such that theMutant Salvador nucleic acid molecule does not encode a functionalSalvador protein. Rather, the Mutant Salvador nucleic acid moleculesencode a Mutant Salvador protein, i.e., a protein which does not exhibitSalvador protein functional activity. In certain preferred embodiments,the Mutant Salvador nucleic acid molecules are truncated forms of SEQ IDNO: 1 or SEQ ID NO:3 which lack one or more WW domains and/or one ormore functional domains.

According to yet another aspect of the invention, isolated nucleic acidmolecules are provided which are based upon the salvador gene genomicsequence (SEQ ID NO:6) and are useful, for example, for detecting thesalvador nucleic acid molecules of the invention. According to thisaspect of the invention, the isolated nucleic acid molecule is selectedfrom:

-   -   (a) nucleic acid molecules which hybridize under stringent        conditions to a nucleic acid molecule having a nucleotide        sequence set forth as SEQ ID NO:6 (human salvador genomic DNA),        and which codes for a Salvador protein,    -   (b) deletions, additions and substitutions of the nucleic acid        molecules of (a), which code for a Salvador protein,    -   (c) nucleic acid molecules that differ from the nucleic acid        molecules of (a) or (b) in codon sequence due to the degeneracy        of the genetic code, and    -   (d) complements of (a), (b) or (c).

The preferred isolated nucleic acids of the invention are salvadornucleic acid molecules which encode a Salvador protein as defined above.

According to yet another aspect of the invention, unique fragments ofthe salvador gene genomic sequence are provided. In this aspect, theisolated nucleic acid molecules are selected from the group consistingof:

-   -   (a) a unique fragment of the nucleotide sequence set forth as        SEQ ID NO:6 between 12 and 2000 nucleotides in length or more        and    -   (b) complements of (a),        wherein the unique fragments exclude nucleic acids having        nucleotide sequences that are contained within SEQ ID NO:6, and        that are known as of the priority date of this application. In        preferred embodiments, the nucleic acid molecule is selected        from the group consisting of: an intron of SEQ ID NO:6, a unique        fragment of an intron of SEQ ID NO:6, and complements of the        foregoing. In a particularly preferred embodiment, the isolated        nucleic acid molecule comprises SEQ ID NO:5.

According to yet another aspect of the invention, an expression vectorcomprising any of the isolated nucleic acid molecules of the inventionoperably linked to a promoter is provided. In a related aspect, hostcells transformed or transfected with such expression vectors also areprovided.

According to still a further aspect of the invention, a transgenicnon-human animal comprising an expression vector of the invention isprovided. Also provided are transgenic non-human animals which havereduced or increased expression of a wild-type salvador nucleic acidmolecule and/or reduced or increased expression of a Mutant salvadornucleic acid molecule. Increased and decreased expression levels aremeasured using methods known to those of skill in art, such as comparingan expression level of a gene or protein of interest in a sample versusa normal expression level, for example, the average expression level ofseveral (e.g., 10) samples.

According to another aspect of the invention, an isolated polypeptideencoded by any of the foregoing isolated nucleic acid molecules of theinvention is provided. Preferably, the isolated polypeptide comprisesSEQ ID NO: 2 or SEQ ID NO: 4. In yet alternative embodiments, theisolated polypeptide comprises a truncated form of SEQ ID NO:2 or SEQ IDNO:4 which lacks one or more WW domains, lacks one or more functional WWdomains, or otherwise includes modifications which result in a proteinthat does not exhibit a Salvador functional activity, i.e., a MutantSalvador protein.

In yet a further aspect of the invention, binding polypeptides thatselectively bind to a salvador molecule, including a wild-type and/orMutant salvador molecule, are provided. According to this aspect, thebinding polypeptides bind to an isolated nucleic acid or protein of theinvention, including binding to unique fragments thereof. Preferably,the binding polypeptides bind to a Salvador protein, a Mutant Salvadorprotein, or a unique fragment of the foregoing. In certain particularlypreferred embodiments, the binding polypeptide binds to a MutantSalvador protein but does not bind to a Salvador protein, i.e., thebinding polypeptides are selective for binding to the Mutant protein andcan be used in various assays to detect the presence of the MutantSalvador protein without detecting the wild type Salvador protein.

In preferred embodiments, the binding polypeptide is an antibody orantibody fragment, more preferably, an Fab or F(ab)₂ fragment of anantibody. Typically, the fragment includes a CDR3 region that isselective for the Salvador protein and/or Mutant Salvador protein. Anyof the various types of antibodies can be used for this purpose,including monoclonal antibodies, humanized antibodies, and chimericantibodies.

According to a further aspect of the invention, pharmaceuticalcompositions containing the nucleic acids, proteins, and bindingpolypeptides of the invention are provided. The pharmaceuticalcompositions contain any of the foregoing salvador molecules, Mutantsalvador molecules, or binding agents in a pharmaceutically acceptablecarrier. Thus, in a related aspect, the invention provides a method forforming a medicament that involves placing a therapeutically effectiveamount of the foregoing agent(s) in the pharmaceutically acceptablecarrier to form one or more doses.

According to another aspect of the invention, various diagnostic methodsare provided. In general, the methods are for diagnosing “a disordercharacterized by aberrant expression of a salvador molecule.” As usedherein, a “disorder characterized by aberrant expression of a salvadormolecule” refers to a disorder in which there is a detectable differencein the expression levels of salvador molecule(s) including wild-typeand/or Mutant salvador molecule(s) in cells that exhibit abnormalcellular development, compared to the expression levels of thesemolecule(s) in cells which do not exhibit such abnormal cellulardevelopment. Abnormal cellular development is determined in accordancewith standard procedures known in the art to assess cell growth, cellmaturation/differentiation, cell proliferation, cell-cell interactions,and cell death (including, e.g., apoptosis). Thus, the observation ofnormal cells is used to establish a control standard for normal cellulardevelopment in a particular cell type.

A disorder characterized by aberrant expression of a salvador moleculeembraces a disorder characterized by underexpression (including nodetectable expression) of a wild-type salvador nucleic acid molecule ora Salvador protein compared to control levels of these molecules (i.e.,levels present in cells which do not exhibit abnormal cellulardevelopment, such as cells obtained from a subject who does not have adisorder characterized by aberrant cellular development), as well asoverexpression of a Mutant salvador nucleic acid molecule or MutantSalvador protein compared to control levels of these molecules. Suchdifferences in expression levels can be determined in accordance withthe diagnostic methods of the invention as disclosed herein. Exemplarycategories of disorders that are characterized by aberrant expression ofa salvador molecule include various cancers, birth defects, andautoimmunity disorders.

As noted above, “aberrant expression” refers to either or both of adecreased expression (including no detectable expression) of thesalvador molecule (nucleic acid or protein) or an increased expressionof a “Mutant salvador molecule.” A Mutant salvador molecule refers to asalvador nucleic acid molecule which includes a mutation (pointmutation, addition, deletion, rearrangement, substitution, truncation,and the like) or to a Salvador protein molecule (e.g., gene product of aMutant salvador nucleic acid molecule) which includes a mutation,provided that the mutation results in a Mutant Salvador protein thatdoes not have the Salvador functional activity that is exhibited by aSalvador protein as described herein. The diagnostic methods of theinvention can be used to detect the presence of a disorder associatedwith aberrant expression of a salvador molecule, as well as to assessthe progression and/or regression of the disorder such as in response totreatment (e.g., chemotherapy, radiation). According to this aspect ofthe invention, the method for diagnosing a disorder characterized byaberrant expression of a salvador molecule involves: detecting in afirst biological sample obtained from a subject, expression of asalvador molecule or a Mutant salvador molecule; wherein decreasedexpression of a salvador molecule or increased expression of a Mutantsalvador molecule compared to a control sample indicates that thesubject has a disorder characterized by aberrant expression of asalvador molecule.

In yet other embodiments, the diagnostic methods are useful fordiagnosing the progression of a disorder. According to theseembodiments, the methods further involve: detecting in a secondbiological sample obtained from the subject, expression of a salvadormolecule or a Mutant salvador molecule, and comparing the expression ofthe salvador molecule or the Mutant salvador molecule in the firstbiological sample and the second biological sample. In theseembodiments, a decrease in the expression of the salvador molecule inthe second biological sample compared to the first biological sample oran increase in the expression of the Mutant salvador molecule in thesecond biological sample compared to the first biological sampleindicates progression of the disorder.

In yet other embodiments, the diagnostic methods are useful fordiagnosing the regression of a disorder. According to these embodiments,the methods further involve: detecting in a second biological sampleobtained from the subject, expression of a salvador molecule or a Mutantsalvador molecule, and comparing the expression of the salvador moleculeor the Mutant salvador molecule in the first biological sample and thesecond biological sample. In these embodiments, an increase in theexpression of the salvador molecule in the second biological samplecompared to the first biological sample or a decrease in the expressionof the Mutant salvador molecule in the second biological sample comparedto the first biological sample indicates regression of the disorder.

In certain embodiments, the diagnostic methods of the invention involvedetecting a salvador molecule that is a salvador nucleic acid moleculeincluding a wild-type and/or Mutant salvador nucleic acid molecule asdescribed above. In yet other embodiments, the methods involve detectinga Salvador protein or Mutant Salvador protein as described above.Various detection methods can be used to practice the diagnostic methodsof the invention. For example, the methods can involve contacting thebiological sample with an agent that selectively binds to the salvadormolecule, to the salvador gene genomic sequence, or to the Mutantsalvador molecule to detect these molecules. In certain embodiments, thesalvador molecule is a nucleic acid and the method involves using anagent that selectively binds to the salvador molecule or to the MutantSalvador molecule, e.g., a nucleic acid that hybridizes to SEQ ID NO:1or to SEQ ID NO:3 under stringent conditions. Alternatively, the methodinvolves using nucleic acids that hybridize under stringent conditionsto a SEQ ID NO:6 that includes the coding sequence of the salvador gene.Such nucleic acid probes include nucleic acids which hybridize to intronportions of the salvador gene (e.g., SEQ ID NO:5). In yet otherembodiments, the salvador molecule is a protein and the method involvesusing an agent that selectively binds to the Salvador protein or to theMutant Salvador protein, e.g., a binding polypeptide, such as anantibody, that selectively binds to SEQ ID NO:2 or to SEQ ID NO:4.

According to still another aspect of the invention, kits for performingthe diagnostic methods of the invention are provided. The kits arenucleic acid-based kits or protein-based kits. According to the formerembodiment, the kits include one or more nucleic acid molecules thathybridize to a salvador nucleic acid molecule including to a wild-typeor Mutant salvador nucleic acid molecule under stringent conditions; oneor more control agents; and instructions for the use of the nucleic acidmolecules, and agents in the diagnosis of a disorder associated withaberrant expression of a salvador molecule. Nucleic acid-based kitsoptionally further include a first primer and a second primer, whereinthe first primer and the second primer are constructed and arranged toselectively amplify at least a portion of a salvador nucleic acidmolecule comprising SEQ ID NO:1 or SEQ ID NO:6 or to selectively amplifya portion of a Mutant salvador nucleic acid molecule. Alternatively, thekits include two isolated nucleic acid molecules, the first consistingof a 20-32 nucleotide contiguous segment of SEQ ID NO:1 or SEQ ID NO:6and the second consisting of a 20-32 nucleotide contiguous segment ofthe complement of SEQ ID NO:1 or SEQ ID NO:6 that does not overlap thefirst segment. Optionally the isolated nucleic acids are uniquefragments of SEQ ID NO:1 or SEQ ID NO:6 or the complements of SEQ IDNO:1 or SEQ ID NO:6. The first and second isolated nucleic acidmolecules are designed to act as primers capable of selectivelyamplifying at least a portion or all of SEQ ID NO:1 or SEQ ID NO:6.Preferably, the nucleic acid molecules that are used to detect and/oramplify the target sequence are selected from the group consisting ofSEQ ID NO:7, SEQ ID NO:8, SEQ D NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, and SEQ ID NO:14.

Alternatively, protein based-kits are provided. Such kits include one ormore binding polypeptides that selectively bind to a Salvador protein orto a Mutant Salvador protein, one or more control agents, andinstructions for the use of the binding polypeptides, and agents in thediagnosis of a disorder associated with aberrant expression of asalvador molecule. In the preferred embodiments, the bindingpolypeptides are antibodies or antigen-binding fragments thereof, suchas those described above. In these and other embodiments, certain of thebinding polypeptides bind to the Mutant Salvador protein but do not bindto the Salvador protein to further distinguish the expression of theseproteins in a biological sample.

The invention also provides treatment methods. In general, the treatmentmethods involve administering an agent to a subject to affect expressionlevels of a salvador molecule, e.g., to increase expression of awild-type salvador molecule and/or reduce expression of a Mutantsalvador molecule, and thereby treat the disorder. Thus, these methodsinclude gene therapy applications. In certain embodiments, the methodfor treating a subject with a disorder characterized by aberrantexpression of a salvador molecule, involves administering to the subjectan effective amount of a salvador nucleic acid molecule to treat thedisorder. In preferred embodiments, the salvador nucleic acid moleculehas SEQ ID NO:1. In other embodiments, the method for treating a subjectwith a disorder characterized by aberrant expression of a salvadormolecule involves administering to the subject an effective amount of aSalvador protein to treat the disorder. In yet other embodiments, thetreatment method involves administering to the subject an effectiveamount of a binding agent (e.g., a nucleic acid or polypeptide) toinhibit a Mutant Salvador molecule and, thereby, treat the disorder. Incertain preferred embodiments, the binding polypeptide is an antibody oran antigen-binding fragment thereof; more preferably, the antibodies orantigen-binding fragments are labeled with one or more cytotoxic agents.Successful treatment of the disorder is determined in accordance withstandard clinical practice for assessing regression of the disorderbeing treated, e.g., a cancer, a birth defect, an autoimmunity disorder.

The invention provides various research methods and compositions. Thus,according to one aspect of the invention, a method for producing aSalvador protein is provided. The method involves providing a salvadornucleic acid molecule operably linked to a promoter, wherein thesalvador nucleic acid molecule encodes the Salvador protein or afragment thereof; expressing the salvador nucleic acid molecule in anexpression system; and isolating the Salvador protein or a fragmentthereof from the expression system. Preferably, the salvador nucleicacid molecule has SEQ ID NO:1 or SEQ ID NO:3. According to yet anotheraspect of the invention, a method for producing a Mutant Salvadorprotein is provided. This method involves: providing a Mutant salvadornucleic acid molecule operably linked to a promoter, wherein the Mutantsalvador nucleic acid molecule encodes the Mutant Salvador protein or afragment thereof; expressing the Mutant salvador nucleic acid moleculein an expression system; and isolating the Mutant Salvador protein or afragment thereof from the expression system. Preferably, the Mutantsalvador nucleic acid molecule has SEQ ID NO:1 or SEQ ID NO:3, with oneor more point mutations, deletions, or truncations, to encode a MutantSalvador protein.

According to other aspects of the invention, various research methodsfor using the compositions of the invention are provided. Exemplaryresearch methods include a method to induce apoptosis in a cell byadministering to the cell an effective amount of a salvador molecule toinduce apoptosis in the cell. Exemplary research methods also include amethod to inhibit cell proliferation in a cell by administering to thecell an effective amount of a salvador molecule to inhibit cellproliferation. Such methods can be performed in vivo or in vitro.

According to yet another aspect of the invention, a method foridentifying a salvador molecule including a Mutant salvador molecule isprovided. The method involves: (a) introducing a putative salvadormolecule or a putative Mutant salvador molecule into a cell; and (b)detecting a salvador functional activity. The salvador functionalactivity is selected from the group consisting of binding to awarts/LATS molecule, modulating cell maturation/differentiation,modulating cell growth, modulating cell proliferation, and modulatingcell death. In certain embodiments, the putative salvador molecule orputative Mutant salvador molecule is derived from a human cell, aDrosophila, or a nematode, e.g., C. elegans.

According to still another aspect of the invention, a method foridentifying a salvador modulating agent that modulates a salvadormolecule-cognate interaction is provided. The method involves (a)contacting a salvador molecule with a salvador molecule cognate, in thepresence of a putative modulating agent, under conditions to allow thesalvador molecule to bind to the cognate, e.g., a warts/LATS molecule,and (b) detecting salvador molecule binding to the cognate. A change insalvador molecule binding to the cognate in the presence of the putativemodulating agent compared to salvador molecule binding to the cognate inthe absence of the cognate indicates that the agent is a modulatingagent of the salvador molecule and/or its cognate. The Salvadormolulating agent may modulate the salvador molecule-cognate interactionby binding to one or both of these binding partners. In general,detecting comprises detecting a change in a parameter selected fromsalvador molecule binding to its cognate, cellmaturation/differentiation, cell growth, cell proliferation, and/or celldeath compared to the parameters detected in the absence of the putativemodulating agent.

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent with reference to the detaileddescription of the preferred embodiments.

Abbreviated Sequence Listing

-   SEQ ID NO:1 is the human Salvador nucleic acid molecule (cDNA).-   SEQ ID NO:2 is the human Salvador protein.-   SEQ ID NO:3 is the Drosophila salvador melanogaster nucleic acid    molecule (cDNA).-   SEQ ID NO: 4 is the Drosophila Salvador protein.-   SEQ ID NO: 5 is a probe for the human salvador gene.-   SEQ ID NO: 6 is the human Salvador gene genomic DNA.-   SEQ ID NO: 7 is a unique fragment of SEQ ID NO:1.-   SEQ ID NO: 8 is a unique fragment of SEQ ID NO:1.-   SEQ ID NO: 9 is a unique fragment of SEQ ID NO:1.-   SEQ ID NO: 10 is a unique fragment of SEQ ID NO:1.-   SEQ ID NO: 11 is a mutant Drosophila Salvador nucleic acid molecule,    Salvador¹.-   SEQ ID NO: 12 is a mutant Drosophila Salvador protein corresponding    to Salvador¹.-   SEQ ID NO: 13 is a mutant Drosophila salvador nucleic acid molecule,    salvador².-   SEQ ID NO: 14 is a mutant Drosophila Salvador protein corresponding    to salvador².-   SEQ ID NO: 15 is a mutant Drosophila Salvador nucleic acid molecule,    salvador³.-   SEQ ID NO: 16 is a mutant Drosophila Salvador protein corresponding    to salvador³.-   SEQ ID NO: 17 is LATS having GenBank accession number U29608.-   SEQ ID NO: 18 is the LATS protein having GenBank accession number    U29608.-   SEQ ID NO: 19 is LATS having GenBank accession number L39837.-   SEQ ID NO: 20 is the LATS protein having GenBank accession number    L39837.-   SEQ ID NO: 21 is LATS1 having GenBank accession number AF104413.-   SEQ ID NO: 22 is the LATS1 protein having GenBank accession number    AF104423.-   SEQ ID NO: 23 is LATS2 having GenBank accession number AB028019.-   SEQ ID NO: 24 is the LATS2 protein having GenBank accession number    AB028019.-   SEQ ID NO: 25 is a peptide used in a protein binding study.-   SEQ ID NO: 26 is a peptide used in a protein binding study.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the Salvador gene organization and protein sequence.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the discovery of a novel Drosophilagene, termed salvador, which is mutated in tumorous outgrowths. Threemutations were identified in the salvador locus that give mutantDrosophila cells a proliferative advantage with respect to wild typecells. The Drosophila salvador gene was cloned and found to encode anovel protein having two WW domains with highly conserved orthologues inC. elegans and mammals. The three salvador mutations identified inDrosophila result in proteins lacking both WW domains.

The two WW domains are located from nucleotide 1350 to 1448 and fromnucleotide 1455 to 1553 of the Drosophila Salvador gene (SEQ ID NO: 3),corresponding to amino acids 424 to 456 and 459 to 491, respectively, ofthe Drosophila Salvador protein (SEQ ID NO: 4).

The human homologue of Salvador, hWW45 (GenBank accession numberNM0218118), has been identified, but its function was previously unknown(Valverde, P., Biochem. Biophys. Res. Commun. 276: 990-998, 2000). Inthe hWW45 gene, the two WW domains are located from nucleotide 813 to911 and from nucleotide 918 to 1016 of SEQ ID NO:1, corresponding toamino acids 200 to 232 and 235 to 267, resepectively, of the humanSalvador protein (SEQ ID NO:2). The Drosophila and human Salvador geneproducts share 47% identity and 54% similarity over the C-termninal 188amino acids.

The invention also is based, in part, on the discovery that the humancounterpart of the Drosophila gene is deleted or mutated in at leastthree tumor-derived cancer cell lines. Accordingly, although not wishingto be bound to any particular theory or mechanism, we believe thesalvador genes from various species play a role in modulating cellulardevelopment (including cell proliferation, growth, and death). Thus, theinvention is directed to novel compositions of the human and Drosophilasalvador nucleic acids and proteins encoded thereby (SEQ ID NOs: 1, 2,3, and 4), as well as to agents that selectively bind to these novelmolecules, and to diagnostic, therapeutic, and research applications ofthese compositions.

According to one aspect of the invention, an isolated nucleic acidmolecule is provided. The isolated nucleic acid molecule is selectedfrom the group consisting of:

-   -   (a) nucleic acid molecules which hybridize under stringent        conditions to a nucleic acid molecule having a nucleotide        sequence set forth as SEQ ID NO:1 or SEQ ID NO:3, and which code        for a Salvador protein,    -   (b) deletions, additions and substitutions of the nucleic acid        molecules of (a), which code for a Salvador protein,    -   (c) nucleic acid molecules that differ from the nucleic acid        molecules of (a) or (b) in codon sequence due to the degeneracy        of the genetic code, and    -   (d) complements of (a), (b), or (c).

The preferred isolated nucleic acids of the invention are salvadornucleic acid molecules which encode a Salvador protein. As used herein,a Salvador protein refers to a protein which is encoded by a nucleicacid having SEQ ID NO:1 or SEQ ID NO:3, or a functional fragmentthereof, or a functional equivalent thereof (e.g., a nucleic acidsequence encoding the same protein as encoded by SEQ ID NO:1 or SEQ IDNO:3), provided that the functional fragment or equivalent encodes aprotein which exhibits a salvador functional activity. As used herein, asalvador functional activity refers to the ability of a salvadormolecule to modulate cellular development such as cellmaturation/differentiation, cell proliferation, growth, and death. Anexemplary salvador functional activity is a tumor suppressor activitywhich includes suppressing and/or reducing tumor cell growth,proliferation, and/or metastasis. Although not wishing to be bound toany particular theory or mechanism, it is believed that the salvadormolecule affects cellular development by promoting cellular arrest inthe G₁ phase of the cell cycle, promoting cell cycle exit, and/orregulating cell death. An assay to measure salvador functional activityis described in the Examples. Specifically, a salvador molecule is saidto have functional activity if the gene product or protein is capable ofdetectably binding to a protein with a PPPY motif.

In the preferred embodiments, the isolated nucleic acid molecule is SEQID NO:1 or SEQ ID NO:3. The invention provides nucleic acid moleculeswhich code for Salvador proteins and which hybridize under stringentconditions to a nucleic acid molecule consisting of the nucleotide setforth in SEQ ID NO:1 or SEQ ID NO:3. Such nucleic acids may be DNA, RNA,or composed of mixed deoxyribonucleotides and ribonucleotides, and mayalso incorporate synthetic non-natural nucleotides. Various methods fordetermining the expression of a nucleic acid and/or a polypeptide innormal and tumor cells are known to those of skill in the art and aredescribed further below and in the Examples. As used herein, the term“protein” is meant to include high molecular weight proteins,polypeptides, low molecular weight peptides, and fragments thereof.

The term “stringent conditions,” as used herein, refers to parameterswith which one skilled in the art is familiar. Nucleic acidhybridization parameters may be found in references which compile suchmethods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, etal., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Morespecifically, stringent conditions, as used herein, refers, for example,to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mMNaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.15Msodium citrate, pH 7; SDS is sodium dodecyl sulphate; and EDTA isethylenediaminetetracetic acid. After hybridization, the membrane uponwhich the DNA is transferred is washed at 2×SSC at room temperature andthen at 0.1×SSC/0.1% SDS at temperatures up to 68° C. The foregoing setof hybridization conditions is but one example of stringenthybridization conditions known to one of ordinary skill in the art.There are other conditions, reagents, and so forth which can be used,which also result in stringent hybridization. The skilled artisan willbe familiar with such conditions, and thus they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of salvador nucleic acid molecules of theinvention. The skilled artisan also is familiar with the methodology forscreening cells and libraries for expression of such molecules whichthen are routinely isolated, followed by isolation of the pertinentnucleic acid molecule and sequencing.

In general, homologs and alleles typically will share at least 35%nucleotide identity and/or at least 40% amino acid identity and/or 50%similarity to SEQ ID NOs:1 or 3 and SEQ ID NOs:2 or 4, respectively, insome instances will share at least 45% nucleotide identity and/or atleast 50% amino acid identity and/or 60% similarity, and in still otherinstances will share at least 55% nucleotide identity and/or at least60%-75% amino acid identity and/or 70%-85% similarity. Preferredhomologs and alleles share nucleotide and amino acid identities with SEQID NO:1 or SEQ ID NO:3 and SEQ ID NO:2 or SEQ ID NO:4, respectively, andencode polypeptides of greater than 70%, more preferably greater than80%, still more preferably greater than 90% and most preferably greaterthan 99% identity. The percent identity can be calculated using various,publicly available software tools developed by NCBI (Bethesda, Md.) thatcan be obtained through the internet (ftp:/ncbi.nlm.nih.gov/pub/).Exemplary tools include the BLAST system, available athttp://www.ncbi.nlm.nih.gov, which uses algorithms developed by Altschulet al. (Nucleic Acids Res. 25:3389-3402, 1997). Pairwise and ClustalWalignments (BLOSUM30 matrix setting) as well as Kyte-Doolittlehydropathic analysis can be obtained using the MacVector sequenceanalysis software (Oxford Molecular Group). Watson-Crick complements ofthe foregoing nucleic acid molecules also are embraced by the invention.

In screening for salvador nucleic acid molecules, a Southern blot may beperformed using the foregoing conditions, together with a radioactiveprobe. After washing the membrane to which the DNA is finallytransferred, the membrane can be placed against X-ray film to detect theradioactive signal.

The invention also includes degenerate nucleic acid molecules whichinclude alternative codons, or triplets, to those present in the nativematerials. For example, serine is encoded by the codons TCA, AGT, TCC,TCG, TCT and AGC. Each of the six codons is equivalent for the purposesof encoding a serine residue. Thus, it will be apparent to one ofordinary skill in the art that any of the serine-encoding nucleotidetriplets may be employed to direct the protein synthesis apparatus, invitro or in vivo, to incorporate a serine residue into a Salvadorprotein. Similarly, nucleotide sequence triplets which encode otheramino acid residues include, but are not limited to: CCA, CCC, CCG andCCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons);ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparaginecodons); and ATA, ATC and ATT (isoleucine codons). Other amino acidresidues may be encoded similarly by multiple nucleotide sequences.Thus, the invention embraces degenerate nucleic acids that differ fromthe biologically isolated nucleic acids in codon sequence due to thedegeneracy of the genetic code.

According to another aspect of the invention, further isolated nucleicacid molecules that are based on the above-noted salvador nucleic acidmolecules are provided. In this aspect, the isolated nucleic acidmolecules are selected from the group consisting of:

-   -   (a) a unique fragment of the nucleotide sequence set forth as        SEQ ID NO:1 or set forth as SEQ ID NO:3 between 12 and 2000        (more preferably, between 12 and 1400 nucleotides in length and,        most preferably, between 12 and 32 nucleotides in length) or        more and    -   (b) complements of (a),    -   wherein the unique fragments exclude nucleic acids having        nucleotide sequences that are contained within SEQ ID NO:1 or        SEQ ID NO:3, and that are known as of the priority date of this        application (Table 1).

Known fragments, i.e., fragments that are not unique, of SEQ ID NO:1 andSEQ ID NO:3 are provided in Table 1 and identified by their positions inSEQ ID NO:1 and SEQ ID NO:3. Overlapping fragments are identified by arange of starting and ending nucleotides (Table 1). For example, afragment which begins with any nucleotide between 1905 and 1916 of SEQ.ID NO:1 and ends with any nucleotide between 1930 and 1955 of SEQ IDNO:1 is not a unique fragment. TABLE 1 Nucleotide sequence containedwithin SEQ ID NO: 1 and SEQ ID NO: 3 that are known as of the prioritydate of this application Starting nucleotide(s) Ending nucleotide(s)Sequence 1 68 SEQ ID NO: 1 38 68 SEQ ID NO: 1 158 309-311 SEQ ID NO: 1174 1707 SEQ ID NO: 1 310 406 SEQ ID NO: 1 749 1021 SEQ ID NO: 1 10191165 SEQ ID NO: 1 1720 1738 SEQ ID NO: 1 1748 2009 SEQ ID NO: 1 17552009 SEQ ID NO: 1 1905-1916 1930-1955 SEQ ID NO: 1 1921-1937 1947-1961SEQ ID NO: 1 1974 1999 SEQ ID NO: 1 1980-1985 1998-2010 SEQ ID NO: 11994 2005 SEQ ID NO: 1 2029 2148-2151 SEQ ID NO: 1 2105 2118 SEQ ID NO:1 2107 2120 SEQ ID NO: 1 2047-2063 2069-2083 SEQ ID NO: 1 1 767 SEQ IDNO: 3 1 892 SEQ ID NO: 3 144 163 SEQ ID NO: 3 155 172 SEQ ID NO: 3 158175 SEQ ID NO: 3 172 190 SEQ ID NO: 3 372 391 SEQ ID NO: 3 386 403 SEQID NO: 3 410-413 431-433 SEQ ID NO: 3 584 603 SEQ ID NO: 3 765-771784-791 SEQ ID NO: 3 773 892 SEQ ID NO: 3 958 1846 SEQ ID NO: 3 11921210 SEQ ID NO: 3 1378 1397 SEQ ID NO: 3 1424 1442 SEQ ID NO: 3 14671484 SEQ ID NO: 3 1528 1547 SEQ ID NO: 3 1812 1829-1830 SEQ ID NO: 31846 2194 SEQ ID NO: 3 1881-1883 1903-1904 SEQ ID NO: 3

Thus, the invention provides isolated unique fragments of SEQ ID NOs:1or 3 or complements of SEQ ID NOs:1 or 3. A unique fragment is one thatis a ‘signature’ for the larger nucleic acid. It, for example, is longenough to assure that its precise sequence is not found in moleculesoutside of the Salvador nucleic acid molecules defined above. Those ofordinary skill in the art may apply no more than routine procedures todetermine if a fragment is unique within the human or Drosophila genome.Unique fragments, however, exclude fragments completely composed of thenucleotide sequences that are contained within SEQ ID NO: 1 or SEQ IDNO: 3 and that are known as of the priority date of this application.

Unique fragments can be used as probes in Southern blot assays toidentify such nucleic acid molecules, or can be used as probes inamplification assays such as those employing the polymerase chainreaction (PCR). As known to those skilled in the art, large probes suchas 200 nucleotides or more are preferred for certain uses such asSouthern blots, while smaller fragments will be preferred for uses suchas PCR. Especially preferred unique fragments include the sequences setforth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. Unique fragmentsalso can be used to produce fusion proteins for generating antibodies ordetermining binding of the polypeptide fragments, or for generatingimmunoassay components. Likewise, unique fragments can be employed toproduce nonfused fragments of the Salvador polypeptides useful, forexample, in the preparation of antibodies, in immunoassays.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of SEQ ID NO:1 and/or SEQ ID NO:3 and its complementwill require longer segments to be unique while others will require onlyshort segments, typically between 12 and 32 nucleotides or more inlength (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, and 32 or more), up to the entire length of thedisclosed sequence. Many segments of the polynucleotide coding region orcomplements thereof that are 18 or more nucleotides in length will beunique. Those skilled in the art are well versed in methods forselecting such sequences, typically on the basis of the ability of theunique fragment to selectively distinguish the sequence of interest fromnon-salvador nucleic acid molecules. A comparison of the sequence of thefragment to those on known data bases typically is all that isnecessary, although in vitro confirmatory hybridization and sequencinganalysis may be performed.

A unique fragment can be a functional fragment. A functional fragment ofa nucleic acid molecule of the invention is a fragment which retainssome functional property of the larger nucleic acid molecule, such ascoding for a functional polypeptide, binding to proteins, regulatingtranscription of operably linked nucleic acid molecules, and the like.One of ordinary skill in the art can readily determine using the assaysdescribed herein and those well known in the art to determine whether afragment is a functional fragment of a nucleic acid molecule using nomore than routine experimentation. Preferred functional fragmentsinclude fragments comprising one or more WW domains of the salvadorgene.

According to yet another aspect of the invention, isolated nucleic acidmolecules that are based on the salvador gene genomic region (SEQ IDNO:6) are provided. According to this aspect of the invention, theisolated nucleic acid molecule is selected from the group consisting of:

-   -   (a) nucleic acid molecules which hybridize under stringent        conditions to a nucleic acid molecule having a nucleotide        sequence set forth as SEQ ID NO:6 (human salvador genomic DNA),        and which codes for a Salvador protein,    -   (b) deletions, additions and substitutions of the nucleic acid        molecules of (a), which code for a Salvador protein,    -   (c) nucleic acid molecules that differ from the nucleic acid        molecules of (a) or (b) in codon sequence due to the degeneracy        of the genetic code, and    -   (d) complements of (a), (b) or (c).        The preferred isolated nucleic acids of the invention are        salvador nucleic acid molecules which encode a Salvador protein        as defined above.

According to yet another aspect of the invention, unique fragments ofthe salvador gene genomic sequence are provided. In this aspect, theisolated nucleic acid molecules are selected from the group consistingof:

-   -   (a) a unique fragment of the nucleotide sequence set forth as        SEQ ID NO:6 between 12 and 2000 nucleotides in length or more        and    -   (b) complements of (a),        wherein the unique fragments exclude nucleic acids having        nucleotide sequences that are contained within. SEQ ID NO:6, and        that are known as of the priority date of this application. In        preferred embodiments, the nucleic acid molecule is selected        from the group consisting of: an exon of SEQ ID NO:6, a unique        fragment of an exon of SEQ ID NO:6, an intron of SEQ ID NO:6, a        unique fragment of an intron of SEQ ID NO:6, and complements of        the foregoing. Exons of SEQ ID NO:6 include nucleotides        3171-3479 (exon 1), nucleotides 5733-6173 (exon 2), nucleotides        25027-25297 (exon 3), nucleotides 27622-27765 (exon 4), and        nucleotides 33131-34873 (exon 5). In a particularly preferred        embodiment, the isolated nucleic acid molecule comprises SEQ ID        NO:5. Preferably, the unique fragments are between 12 and 2000        nucleotides in length, more preferably between 12 and 1400        nucleotides in length.

In yet another aspect of the invention, Mutant salvador nucleic acidmolecules are provided. The Mutant salvador nucleic acid moleculescontain a sequence which is identical to SEQ ID NO:1 or SEQ ID NO:3,with the exception that the sequence includes one or more mutations,e.g., deletions, additions, substitutions, or truncations, such that theMutant salvador nucleic acid molecule does not encode a functionalSalvador protein. Rather, the Mutant salvador nucleic acid moleculesencode a Mutant Salvador protein, i.e., a protein which does not exhibita Salvador protein functional activity. Typically, there are fewer than30 deletions, additions, substitutions, truncations, or combinationsthereof. In some embodiments, there are 30, 29, 28, 27, 26, 25, 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 mutation(s).

Three Mutant salvador nucleic acid molecules were identified inDrosophila using a genetic screen, as described in the Examples. TheMutant salvador nucleic acid sequences salvador¹, salvador², andsalvador³ are set forth in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO:15, respectively. The predicted sequences of Mutant proteinscorresponding to the foregoing genes are set forth in SEQ ID NO: 12, SEQID NO: 14, and SEQ ID NO: 16 respectively. These four Mutant salvadorgenes include mutations which prohibit at least nucleotides 1350-1448and 1455-1553 of SEQ ID NO:3 from being expressed, resulting in MutantSalvador protein sequences that are truncated or otherwise mutatedproteins lacking both WW domains characteristic of WW domain proteins.Preferred human Mutant salvador nucleic acid molecules have a nucleicacid sequence that is a truncated form of SEQ ID NO:1. These Mutantsalvador nucleic acid molecules result in Mutant human Salvador proteinslacking one or more WW domains, which are located between amino acids200 to 232 and 235 to 267 of SEQ ID NO:2.

As used herein, with respect to nucleic acid molecules, the term“isolated” means: (i) amplified in vitro by, for example, PCR; (ii)recombinantly produced by cloning; (iii) purified, for example, bycleavage and gel separation; or (iv) synthesized, for example,chemically. An isolated nucleic acid molecule is one which is readilymanipulable by recombinant DNA techniques well known in the art. Thus, anucleotide sequence contained in a vector in which 5′ and 3′ restrictionsites are known or for which PCR primer sequences have been disclosed isconsidered isolated but a nucleic-acid sequence existing in its nativestate in its natural host is not. An isolated nucleic acid molecule maybe substantially purified, but need not be. For example, a nucleic acidmolecule that is isolated within a cloning or expression vector is notpure in that it may comprise only a tiny percentage of the material inthe cell in which it resides. Such a nucleic acid molecule is isolated,however, as the term is used herein, because it is readily manipulableby standard techniques known to those of ordinary skill in the art. Anisolated nucleic acid molecule, as used herein, does not include anaturally occurring chromosome.

As used herein, a “Mutant Salvador nucleic acid molecule” refers to aSalvador nucleic acid molecule which includes a mutation (pointmutation, addition, deletion, rearrangement, substitution, truncation,and the like) such that the Mutant salvador nucleic acid molecule doesnot encode a functional Salvador protein. Rather, the Mutant salvadornucleic acid molecule encodes a Mutant Salvador protein, i.e., a proteinwhich does not exhibit a Salvador protein functional activity. A “MutantSalvador protein” refers to a Salvador protein that is a protein productof a Mutant salvador nucleic acid molecule which includes a mutationthat affects the functional activity of the salvador molecule. PreferredMutant Salvador proteins are those which lack one or more WW domainsand/or one or more functional WW domains, e.g., the protein can be atruncated form of the Salvador protein having SEQ ID NO:2.

As used herein, the term “aberrant expression” refers to either or bothof decreased expression (including no detectable expression) of asalvador molecule (nucleic acid or protein) or increased expression of aMutant salvador molecule (nucleic acid or protein). The term“pharmaceutically acceptable” means a non-toxic material that does notinterfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

According to yet another aspect of the invention, an expression vectorcomprising any of the isolated nucleic acid molecules of the invention,preferably operably linked to a promoter is provided. In a relatedaspect, host cells transformed or transfected with such expressionvectors also are provided.

As used herein, a “vector” may be any of a number of nucleic acidmolecules into which a desired sequence may be inserted by restrictionand ligation for transport between different genetic environments or forexpression in a host cell. Vectors are typically composed of DNAalthough RNA vectors are also available. Vectors include, but are notlimited to, plasmids, phagemids, and virus genomes. A cloning vector isone which is able to replicate in a host cell, and which is furthercharacterized by one or more endonuclease restriction sites at which thevector may be cut in a determinable fashion and into which a desired DNAsequence may be ligated such that the new recombinant vector retains itsability to replicate in the host cell. In the case of plasmids,replication of the desired sequence may occur many times as the plasmidincreases in copy number within the host bacterium or just a single timeper host before the host reproduces by mitosis. In the case of phage,replication may occur actively during a lytic phase or passively duringa lysogenic phase. An expression vector is one into which a desired DNAsequence may be inserted by restriction and ligation such that it isoperably joined to regulatory sequences and may be expressed as an RNAtranscript. Vectors may further contain one or more marker sequencessuitable for use in the identification of cells which have or have notbeen transformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art, e.g., β-galactosidase or alkaline phosphatase, and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques, e.g., green fluorescent protein. Preferredvectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which theyare operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. As used herein,“operably joined” and “operably linked” are used interchangeably andshould be construed to have the same meaning. If it is desired that thecoding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Often, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

It will also be recognized that the invention embraces the use of thesalvador cDNA and genomic sequences or Mutant Salvador cDNA and genomicsequences in expression vectors, as well as to transfect host cells andcell lines, be these prokaryotic, e.g., E. coli or eukaryotic, e.g., CHOcells, COS cells, yeast expression systems, and recombinant baculovirusexpression in insect cells. Especially useful are mammalian cells suchas human, mouse, hamster, pig, goat, primate, etc. They may be of a widevariety of tissue types, including mast cells, fibroblasts, oocytes, andlymphocytes, and may be primary cells and cell lines. Specific examplesinclude dendritic cells, U293 cells keratinocytes, peripheral bloodleukocytes, bone marrow stem cells and embryonic stem cells. Theexpression vectors require that the pertinent sequence, i.e., thosenucleic acids described supra, be operably linked to a promoter.

According to still a further aspect of the invention, a transgenicnon-human animal comprising an expression vector of the invention isprovided, including a transgenic non-human animal which has reduced orincreased expression of a salvador nucleic acid molecule or elevatedexpression of a Mutant salvador nucleic acid molecule.

As used herein, the term “transgenic non-human animals” includesnon-human animals having one or more exogenous nucleic acid moleculesincorporated in germ line cells and/or somatic cells. Thus, thetransgenic animals include “knockout” animals having a homozygous orheterozygous gene disruption by homologous recombination, animals havingepisomal or chromosomally incorporated expression vectors, etc. Knockoutanimals can be prepared by homologous recombination using embryonic stemcells as is well known in the art. The recombination can be facilitatedby the cre/lox system or other recombinase systems known to one ofordinary skill in the art. In certain embodiments, the recombinasesystem itself is expressed conditionally, for example, in certaintissues or cell types, at certain embryonic or post-embryonicdevelopmental stages, inducibly by the addition of a compound whichincreases or decreases expression, and the like. In general, theconditional expression vectors used in such systems use a variety ofpromoters which confer the desired gene expression pattern, e.g.,temporal or spatial patterns. Conditional promoters also can be operablylinked to salvador nucleic acid molecules to increase or decreaseexpression of a salvador molecule in a regulated or conditional manner.Trans-acting negative or positive regulators of salvador activity orexpression also can be operably linked to a conditional promoter asdescribed above. Such trans-acting regulators include antisense salvadornucleic acid molecules, nucleic acid molecules which encode dominantnegative salvador molecules, ribozyme molecules specific for salvadornucleic acid molecules, and the like. The transgenic non-human animalsare useful in experiments directed toward testing biochemical orphysiological effects of diagnostics or therapeutics for conditionscharacterized by increased or decreased salvador molecule expression.Other uses will be apparent to one of ordinary skill in the art. Thus,the invention also permits the construction of salvador gene “knockouts”in cells and in animals, providing materials for studying certainaspects of cellular development including, e.g., modulating cellmaturation/differentiation, cell growth, cell proliferation, and celldeath, as well as metastasis.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA or RNA encoding a Salvador protein, a Mutant Salvadorprotein, fragments, or variants thereof. The heterologous DNA or RNA isplaced under operable control of transcriptional elements to permit theexpression of the heterologous DNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen), which contains an Epstein Barr virus (EBV) originof replication, facilitating the maintenance of plasmid as a multicopyextrachromosomal element. Another expression vector is the pEF-BOSplasmid containing the promoter of polypeptide Elongation Factor 1α,which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mizushima and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant isdescribed by Warnier et al., in intradermal injection in mice forimmunization against P1A (Int. J. Cancer, 67:303-310, 1996).

The invention also embraces kits termed expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

According to another aspect of the invention, an isolated proteinencoded by any of the foregoing isolated nucleic acid molecules of theinvention is provided. Preferably, the isolated protein comprises SEQ IDNO: 2 or SEQ ID NO: 4, for example, fragments comprising at least one WWdomain. The invention also embraces Mutant Salvador proteins, such asthose described in the Examples and other Mutant Salvador proteins suchas truncated forms of SEQ ID NO:2 which lack one or more functional WWdomains.

The invention also provides isolated proteins, which include theproteins of SEQ ID NOs:2 and 4 and unique fragments of SEQ ID NOs:2 and4, for example, fragments comprising at least one functional WW domain.Such proteins are useful, for example, alone or as fusion proteins togenerate antibodies (for example, as therapeutics), or as a component(s)of an immunoassay.

As used herein, a Salvador protein refers to a protein which is encodedby a nucleic acid having SEQ ID NO:1 or SEQ ID NO:3, a functionalfragment thereof, or a functional equivalent thereof (e.g., a nucleicacid sequence encoding the same protein as encoded by SEQ ID NO:1 or SEQID NO:3), provided that the functional fragment or equivalent encodes aSalvador protein which exhibits a salvador functional activity. As usedherein, a “salvador functional activity” refers to the ability of aSalvador protein to modulate cellular development, e.g., cellmaturation/differentiation, cell growth, cell proliferation, and celldeath. Modulating cellular development can be detected usingconventional assays which detect any one or more of cellmaturation/differentiation, cell growth, proliferation (including, e.g.,cell metastasis), and death (including, e.g., apoptosis assays). Thus,an exemplary salvador functional activity is a tumor suppressor activitysuch as suppressing and/or reducing tumor cell growth, proliferation,and/or metastasis. Although not wishing to be bound to any particulartheory or mechanism, it is believed that the Salvador protein modulatesat least some of the above-noted cell functions by promoting cellulararrest in the G₁ phase of the cell cycle, promoting cell cycle exit,and/or regulating cell death.

Proteins can be isolated from biological samples including tissue orcell homogenates, and can also be expressed recombinantly in a varietyof prokaryotic and eukaryotic expression systems by constructing anexpression vector appropriate to the expression system, introducing theexpression vector into the expression system, and isolating therecombinantly expressed protein. Short polypeptides, including antigenicpeptides (such as are presented by MHC molecules on cell surfaces forimmune recognition) also can be synthesized chemically usingwell-established methods of peptide synthesis.

Thus, as used herein with respect to proteins, “isolated” meansseparated from its native environment and present in sufficient quantityto permit its identification or use. Isolated, when referring to aprotein or polypeptide, means, for example: (i) selectively produced byexpression of a recombinant nucleic acid or (ii) purified as bychromatography or electrophoresis. Isolated proteins or polypeptidesmay, but need not be, substantially pure. The term “substantially pure”means that the proteins or polypeptides are essentially free of othersubstances with which they may be found in nature or in vivo systems toan extent practical and appropriate for their intended use.Substantially pure proteins may be produced by techniques well known inthe art. Because an isolated protein may be admixed with apharmaceutically acceptable carrier in a pharmaceutical preparation, theprotein may comprise only a small percentage by weight of thepreparation. The protein is nonetheless isolated in that it has beenseparated from the substances with which it may be associated in livingsystems, e.g., isolated from other proteins.

A fragment of a Salvador protein, for example, generally has thefeatures and characteristics of fragments including unique fragments asdiscussed above in connection with nucleic acid molecules. As will berecognized by those skilled in the art, the size of a fragment which isunique will depend upon factors such as whether the fragment constitutesa portion of a conserved protein domain. Thus, some regions of Salvadorproteins will require longer segments to be unique while others willrequire only short segments, typically between 5 and 12 amino acids(e.g., 5, 6, 7, 8, 9, 10, 11, and 12 amino acids long).

Unique fragments of a protein preferably are those fragments whichretain a distinct functional capability of the protein. Functionalcapabilities which can be retained in a fragment of a protein includeinteraction with antibodies, interaction with other proteins orfragments thereof, selective binding of nucleic acid molecules, andenzymatic activity. One important activity is the ability to act as asignature for identifying the polypeptide. Another is the ability toprovoke in an animal an immune response to a Mutant salvador moleculebut not provoke an immune response to a salvador molecule and, thereby,create antibodies that are selective for the Mutant salvador molecule.

Those skilled in the art are well versed in methods for selectingfragments with unique amino acid sequences, typically on the basis ofthe ability of the fragment to selectively distinguish the sequence ofinterest from non-family members. A comparison of the sequence of thefragment to those in known data bases typically is all that isnecessary.

The invention embraces variants and mutants of the Salvador proteinsdescribed herein. As used herein, a “variant” of a Salvador protein is aprotein which contains one or more modifications to the primary aminoacid sequence of a Salvador protein. Modifications which create aSalvador protein variant can be made to a Salvador protein (1) toproduce, increase, reduce, or eliminate an activity of the Salvadorprotein; (2) to enhance a property of the Salvador protein, such asprotein stability in an expression system or the stability ofprotein-protein binding; or (3) to provide a novel activity or propertyto a Salvador protein, such as addition of an antigenic epitope oraddition of a detectable moiety. Modifications to a Salvador protein orto a Mutant Salvador protein are typically made to the nucleic acidmolecule which encodes the protein, and can include deletions, pointmutations, truncations, rearrangements, amino acid substitutions, andadditions of amino acids or non-amino acid moieties.

Mutations of a nucleic acid molecule which encode a Salvador proteinpreferably preserve the amino acid reading frame of the coding sequence,and preferably do not create regions in the nucleic acid which arelikely to hybridize to form secondary structures, such as hairpins orloops, which can be deleterious to expression of the variant protein.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe protein. Variant proteins are then expressed and tested for one ormore activities to determine which mutation provides a variant proteinwith the desired properties. Further mutations can be made to variants(or to non-variant Salvador proteins) which are silent as to the aminoacid sequence of the protein, but which provide preferred codons fortranslation in a particular host. The preferred codons for translationof a nucleic acid in, e.g., E. coli are well known to those of ordinaryskill in the art. Still other mutations can be made to the noncodingsequences of a salvador gene or cDNA clone to enhance expression of theprotein. The activity of variants of Salvador proteins can be tested bycloning the gene encoding the variant Salvador protein into a bacterialor mammalian expression vector, introducing the vector into anappropriate host cell, expressing the variant Salvador protein, andtesting for a functional capability of the Salvador protein as disclosedherein.

Alternatively, modifications can be made directly to the Salvadorprotein, such as by cleavage, addition of a linker molecule, addition ofa detectable moiety, such as biotin, addition of a fatty acid, and thelike. Modifications also embrace fusion proteins comprising all or partof the salvador amino acid sequences, or proteins which retain the sameconformation and/or function but have a designed sequence. One of skillin the art will be familiar with methods for designing a proteinsequence for a particular protein conformation, and can thus “design” avariant Salvador polypeptide according to known methods. One example ofsuch a method is described by Dahiyat and Mayo in Science 278:82-87,1997, and U.S. Patent Nos. 6,188,965 and 6,269,312, whereby proteins orportions thereof can be designed de novo. These methods can also beapplied to a known protein to vary only a portion of the proteinsequence. By applying computational methods, specific variants of aSalvador protein can be proposed and tested to determine whether thevariant retains a desired conformation and/or function. In general,variants include Salvador proteins which are modified specifically toalter a feature of the protein unrelated to its desired physiologicalactivity. For example, cysteine residues can be substituted or deletedto prevent unwanted disulfide linkages. Similarly, certain amino acidscan be changed to enhance expression of a Salvador protein byeliminating proteolysis by proteases in an expression system, e.g.,dibasic amino acid residues in yeast expression systems in which KEX2protease activity is present. Additionally, some or all of the proteinmay be redesigned to, for example, enhance stability or shelf life.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in Salvador proteins to provide functionalvariants of the foregoing proteins, i.e., the variants retain thefunctional capabilities of the Salvador proteins. As used herein, a“conservative amino acid substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Conservative substitutions of amino acids include substitutionsmade amongst amino acids within the following groups: (a) M, 1, L, V;(b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

For example, upon determining that a peptide derived from a Salvadorprotein plays a role in cellular development, e.g., tumor suppression,metastasis, cell maturation/differentiation, cell growth, cellproliferation, cell death, and/or apoptosis, one can make conservativeamino acid substitutions to the amino acid sequence of the peptide. Thesubstituted peptides can then be tested for one or more of theabove-noted functions, in vivo or in vitro. These variants can be testedfor improved stability and are useful, inter alia, in pharmaceuticalcompositions.

Functional variants of Salvador proteins, i.e., variants of proteinswhich retain the function of the Salvador proteins, can be preparedaccording to methods for altering polypeptide sequence known to one ofordinary skill in the art such as are found in references which compilesuch methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplaryfunctional variants of the Salvador proteins include conservative aminoacid substitutions of proteins encoded by SEQ ID NOs:2 or 4.Conservative amino acid substitutions in the amino acid sequence ofSalvador proteins to produce functional variants of Salvador proteinstypically are made by alteration of the nucleic acid molecule encoding aSalvador protein (e.g., SEQ ID NO:1 or SEQ ID NO:3). Such substitutionscan be made by a variety of methods known to one of ordinary skill inthe art. For example, amino acid substitutions may be made byPCR-directed mutations, site-directed mutagenesis according to themethod of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,1985), or by chemical synthesis of a gene encoding a Salvador protein.Where amino acid substitutions are made to a small unique fragment of aSalvador protein, the substitutions can be made by directly synthesizingthe peptide. The activity of functional variants or fragments ofSalvador protein can be tested by cloning the gene encoding the alteredSalvador protein into a bacterial or mammalian expression vector,introducing the vector into an appropriate host cell, expressing thealtered Salvador protein, and testing for a functional capability of theSalvador protein as disclosed herein.

The invention as described herein has a number of uses, some of whichare described elsewhere herein. First, the invention permits isolationof the Salvador proteins and Mutant Salvador proteins. A variety ofmethodologies well-known to the skilled practitioner can be utilized toobtain isolated Salvador proteins and Mutant Salvador proteins. Theproteins may be purified from cells which naturally produce the proteinby chromatographic means or immunological recognition. Alternatively, anexpression vector may be introduced into cells to cause production ofthe protein. In another method, mRNA transcripts may be microinjected orotherwise introduced into cells to cause production of the encodedprotein. Translation of mRNA in cell-free extracts such as thereticulocyte lysate system also may be used to produce the protein.Those skilled in the art also can readily follow known methods forisolating Salvador proteins and Mutant Salvador proteins. These include,but are not limited to, chromatographic techniques such asimmunochromatography, HPLC, size-exclusion chromatography, ion-exchangechromatography, and immune-affinity chromatography.

The isolation and identification of salvador nucleic acid molecules andof Mutant salvador nucleic acid molecules also allows one of skill inthe art to diagnose a disorder characterized by aberrant expression of asalvador nucleic acid molecule or protein or of a Mutant salvadornucleic acid molecule or protein. These methods involve determining theaberrant expression of one or more Salvador nucleic acid moleculesand/or Mutant Salvador nucleic acid molecules, and/or encoded Salvadorproteins and/or Mutant Salvador proteins. In the former two situations,such determinations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction, orassaying with hybridization probes which may be labeled. In the lattertwo situations, such determinations can be carried out by assayingbiological samples with binding partners (e.g., antibodies) for Salvadorproteins or Mutant Salvador proteins.

The invention also provides, in certain embodiments, “dominant negative”polypeptides derived from Salvador proteins and/or Mutant Salvadorproteins. A dominant negative polypeptide is an inactive variant of aprotein, which, by interacting with the cellular machinery, displaces anactive protein from its interaction with the cellular machinery orcompetes with the active protein, thereby reducing the effect of theactive protein. Dominant negative polypeptides are useful, or example,for preparing transgenic non-human animals to further characterize thefunctions of the salvador molecules and Mutant salvador moleculesdisclosed herein. For example, a dominant negative receptor which bindsa ligand but does not transmit a signal in response to binding of theligand can reduce the biological effect of expression of the ligand.Likewise, a dominant negative catalytically-inactive kinase whichinteracts normally with target proteins but does not phosphorylate thetarget proteins can reduce phosphorylation of the target proteins inresponse to a cellular signal. Similarly, a dominant negativetranscription factor which binds to a promoter site in the controlregion of a gene but does not increase gene transcription can reduce theeffect of a normal transcription factor by occupying promoter bindingsites without increasing transcription.

The end result of the expression of a dominant negative polypeptide in acell is a reduction in function of active proteins. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a protein, and using standard mutagenesis techniques tocreate one or more dominant negative variant polypeptides. For example,one of ordinary skill in the art can modify the sequence of Salvadorproteins by site-specific mutagenesis, scanning mutagenesis, partialgene deletion or truncation, and the like. See, e.g., U.S. Pat. No.5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, 1989. The skilledartisan then can test the population of mutagenized proteins fordiminution in a selected and/or for retention of such an activity. Othersimilar methods for creating and testing dominant negative variants of aprotein will be apparent to one of ordinary skill in the art.

In yet a further aspect of the invention, binding polypeptides thatselectively bind to a salvador molecule and/or to a Mutant salvadormolecule are provided. According to this aspect, the bindingpolypeptides bind to an isolated nucleic acid or protein of theinvention, including unique fragments thereof. Preferably, the bindingpolypeptides bind to a Salvador protein, a Mutant Salvador protein, or aunique fragment thereof. In certain particularly preferred embodiments,the binding polypeptide binds to a Mutant Salvador protein but does notbind to a Salvador protein, i.e., the binding polypeptides are selectivefor binding to the Mutant Salvador protein and can be used in variousassays to detect the presence of the Mutant Salvador protein withoutdetecting Salvador protein. Such Mutant Salvador protein bindingpolypeptides also can be used to selectively bind to a Mutant salvadormolecule in a cell (in vivo or ex vivo) for imaging and therapeuticapplications in which, for example, the binding polypeptide is taggedwith a detectable label and/or a toxin for targeted delivery to thesalvador molecule. Of course, other binding polypeptides can bedeveloped which bind selectively to the Salvador protein and not to theMutant Salvador protein to, for example, selectively identify expressionof a Salvador protein in a cell.

In preferred embodiments, the binding polypeptide is an antibody orantibody fragment, more preferably, an Fab or F(ab)₂ fragment of anantibody. Typically, the fragment includes a CDR3 region that isselective for the Salvador protein and/or Mutant Salvador protein. Anyof the various types of antibodies can be used for this purpose,including polyclonal antibodies, monoclonal antibodies, humanizedantibodies, and chimeric antibodies.

Thus, the invention provides agents which bind to Salvador proteinsand/or Mutant Salvador proteins encoded by salvador nucleic acidmolecules and/or Mutant salvador nucleic acid molecules, respectively,and in certain embodiments preferably to unique fragments of theSalvador proteins or Mutant Salvador proteins. Such binding partners canbe used in screening assays to detect the presence or absence of aSalvador protein and/or a Mutant Salvador protein and in purificationprotocols to isolate such Salvador proteins and/or Mutant Salvadorproteins. Likewise, such binding partners can be used to selectivelytarget drugs, toxins or other molecules to cells which express Salvadorand/or Mutant Salvador proteins. In this manner, for example, cellspresent in solid or non-solid tumors which express Mutant Salvadorproteins can be treated with cytotoxic compounds that are selective forthe Mutant Salvador molecules (nucleic acids and/or proteins). Suchbinding agents also can be used to inhibit the native activity of theSalvador and/or Mutant Salvador proteins, for example, by binding tosuch proteins, to further characterize the functions of these molecules.

The invention, therefore, provides antibodies or fragments of antibodieshaving the ability to selectively bind to Salvador proteins and/orMutant Salvador proteins, and preferably to unique fragments of theforegoing. Antibodies include polyclonal, monoclonal, and chimericantibodies, prepared, e.g., according to conventional methodology.

The antibodies of the present invention thus are prepared by any of avariety of methods, including administering a protein, fragments of aprotein, cells expressing the protein or fragments thereof and the liketo an animal to induce polygonal antibodies. The production ofmonoclonal antibodies is according to techniques well known in the art.As detailed herein, such antibodies may be used for example to identifytissues expressing protein or to purify protein. Antibodies also may becoupled to specific labeling agents for imaging or to antitumor agents,including, but not limited to, methotrexate, radioiodinated compounds,toxins such as ricin, other cytostatic or cytolytic drugs, and so forth.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Fab fragments consist of acovalently bound antibody light chain and a portion of the antibodyheavy chain denoted Fd. The Fd fragments are the major determinant ofantibody specificity (a single Fd fragment may be associated with up toten different light chains without altering antibody specificity) and Fdfragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of nonspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv, and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies. Thus, the invention involves polypeptides ofnumerous size and type that bind specifically to Salvador proteinsand/or Mutant Salvador proteins. These polypeptides may be derived alsofrom sources other than antibody technology. For example, suchpolypeptide binding agents can be provided by degenerate peptidelibraries which can be readily prepared in solution, in immobilized formor as phage display libraries. Combinatorial libraries also can besynthesized of peptides containing one or more amino acids. Librariesfurther can be synthesized of peptides and non-peptide syntheticmoieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using, e.g., m13, fd, or lambda phage), displayinginserts from 4 to about 80 amino acid residues using conventionalprocedures. The inserts may represent a completely degenerate or biasedarray. One then can select phage-bearing inserts which bind to aSalvador protein or a Mutant Salvador protein. This process can berepeated through several cycles of reselection of phage that bind to aSalvador protein and/or a Mutant Salvador protein. Repeated rounds leadto enrichment of phage bearing particular sequences. DNA sequenceanalysis can be conducted to identify the sequences of the expressedpolypeptides. The minimal linear portion of the sequence that binds tothe Salvador protein and/or to the Mutant Salvador protein can bedetermined. One can repeat the procedure using a biased librarycontaining inserts containing part or all of the minimal linear portionplus one or more additional degenerate residues upstream or downstreamthereof. Thus, the Salvador proteins of the invention can be used toscreen peptide libraries, including phage display libraries, to identifyand select peptide binding partners of the Salvador proteins and/orMutant Salvador proteins of the invention. Such molecules can be used,as described, for screening assays, for diagnostic assays, forpurification protocols or for targeting drugs, toxins and/or labelingagents (e.g., radioisotopes, fluorescent molecules, etc.) to cells whichexpress Mutant salvador genes such as cancer cells which have aberrantsalvador expression. As detailed herein, the foregoing antibodies andother binding molecules may be used to identify tissues with normal oraberrant expression of a Salvador protein, for example, to identifytissues expressing abnormal levels of a Salvador protein and/or of aMutant Salvador protein, compared to the expression of these proteins intissues which express a normal level of these proteins (i.e., levels ofSalvador and/or Mutant Salvador protein in tissues which are notcharacterized by tumor cell growth and/or metastasis or other abnormalcell maturation/differentiation, abnormal cell growth, abnormal cellproliferation, abnormal cell death, and/or abnormal cell apoptosis.Additionally, the binding molecules may be used to purify Salvadorproteins or Mutant Salvador proteins.

Antibodies also may be coupled to specific diagnostic labeling agentsfor imaging of cells and tissues with normal or aberrant salvadorexpression or to therapeutically useful agents according to standardcoupling procedures. Diagnostic agents include, but are not limited to,barium sulfate, iocetamic acid, iopanoic acid, ipodate calcium,diatrizoate sodium, diatrizoate meglumine, metrizamide, tyropanoatesodium and radiodiagnostics including positron emitters such asfluorine-18 and carbon-11, gamma emitters such as iodine-123,technitium-99, iodine-131 and indium-111, and nuclides for nuclearmagnetic resonance such as fluorine and gadolinium. Other diagnosticagents useful in the invention will be apparent to one of ordinary skillin the art.

As used herein, “therapeutically useful agents” include any therapeuticmolecule known in the art. These agents include antineoplastic agents,radioiodinated compounds, toxins, other cytostatic or cytolytic drugs,and so forth. Antineoplastic therapeutics are well known and include:aminoglutethimide, azathioprine, bleomycin sulfate, busulfan,carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine,cytarabidine, dacarbazine, dactinomycin, daunorubicin, doxorubicin,taxol, etoposide, fluorouracil, interferon, lomustine, mercaptopurine,methotrexate, mitotane, procarbazine HCl, thioguanine, vinblastinesulfate and vincristine sulfate. Additional antineoplastic agentsinclude those disclosed in Chapter 52, Antineoplastic Agents (PaulCalabresi and Bruce A. Chabner), and the introduction thereto, pp.1202-1263, of Goodman and Gilman's “The Pharmacological Basis ofTherapeutics,” Eighth Edition, 1990, McGraw-Hill, Inc. (HealthProfessions Division). Toxins can be proteins such as, for example,pokeweed anti-viral protein, cholera toxin, pertussis toxin, ricin,gelonin, abrin, diphtheria exotoxin, or Pseudomonas exotoxin. Toxinmoieties can also be high energy-emitting radionuclides such ascobalt-60. In one embodiment, these agents may be targeted selectivelyto a cell or tissue selectively with an aberrant salvador expression.

According to a further aspect of the invention, pharmaceuticalcompositions containing the nucleic acid molecules, proteins, andbinding polypeptides of the invention are provided. The pharmaceuticalcompositions contain any of the foregoing molecules of the invention ina pharmaceutically acceptable carrier. Thus, in a related aspect, theinvention provides a method for forming a medicament that involvesplacing a therapeutically effective amount of a molecule of theinvention in the pharmaceutically acceptable carrier to form one or moredoses.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines, and optionally other therapeutic agents.

As used herein, the term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients. The term “physiologicallyacceptable” refers to a non-toxic material that is compatible with abiological system such as a cell, cell culture, tissue, or organism. Thecharacteristics of the carrier will depend on the route ofadministration. Physiologically and pharmaceutically acceptable carriersinclude diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials which are well known in the art.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712). Those of skill in the art can readilydetermine the various parameters and conditions for producing antibodyaerosols without undue experimentation.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, and lactated Ringer's or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases, and the like.

The preparations of the invention are administered in effective amounts.As used herein, an “effective amount” is that amount of a pharmaceuticalpreparation that alone, or together with further doses, stimulates adesired response. In the case of treating cancer, the desired responseis inhibiting the progression of the cancer. This may involve onlyslowing the progression of the disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently. In the case of stimulating an immune response (e.g., forresearch applications to prepare antibodies or for therapeuticapplications to induce antibodies to a Mutant salvador molecule but notto a salvador molecule), the desired response is an increase inantibodies or T lymphocytes which are specific for the immunogen(s)employed. These responses can be monitored by routine methods or can bemonitored according to diagnostic methods of the invention discussedherein.

Where it is desired to stimulate an immune response using a therapeuticcomposition of the invention (e.g., a Mutant Salvador protein fragmentwhich is a unique fragment of the Mutant salvador molecule and is absentfrom the Salvador protein), a desired response may involve thestimulation of a humoral antibody response resulting in an increase inantibody titer in serum, a clonal expansion of cytotoxic lymphocytes, orsome other desirable immunologic response. It is believed that doses ofimmunogens ranging from one nanogram/kilogram to 100milligrams/kilogram, depending upon the mode of administration, would beeffective. The preferred range is believed to be between 500 nanogramsand 500 micrograms per kilogram. The absolute amount will depend upon avariety of factors, including the material selected for administration,whether the administration is in single or multiple doses, andindividual patient parameters including age, physical condition, size,weight, and the stage of the disease. These factors are well known tothose of ordinary skill in the art and can be addressed with no morethan routine experimentation.

According to another aspect of the invention, various diagnostic methodsare provided. In general, the methods are for diagnosing a disordercharacterized by aberrant expression of a salvador molecule or a Mutantsalvador molecule. As used herein, “aberrant expression” refers toeither or both of a decreased expression (including no detectableexpression) of a Salvador molecule (nucleic acid or protein) or anincreased expression of a “Mutant salvador molecule”. A Mutant Salvadormolecule refers to a Salvador nucleic acid molecule which includes amutation (point, deletion, addition, substitution, rearrangement,truncation) or to a Mutant Salvador protein molecule (e.g., gene productof Mutant Salvador nucleic acid molecule) which includes a mutation,provided that the mutation results in a Mutant Salvador protein thatdoes not have a Salvador protein functional activity. The diagnosticmethods of the invention can be used to detect the presence of adisorder associated with aberrant expression of a Salvador molecule or aMutant salvador molecule, as well as to assess the progression and/orregression of the disorder such as in response to treatment (e.g.,chemotherapy, radiation).

According to this aspect of the invention, the method for diagnosing adisorder characterized by aberrant expression of a Salvador molecule ora Mutant salvador molecule involves: detecting in a first biologicalsample obtained from a subject, expression of a salvador molecule or aMutant salvador molecule; wherein decreased expression of a salvadormolecule or increased expression of a Mutant salvador molecule comparedto a control sample indicates that the subject has a disordercharacterized by aberrant expression of a salvador molecule.

In one embodiment, a probe sequence is used to screen for aberrantexpression of a salvador molecule. In a preferred embodiment, the probehas the sequence set forth as SEQ ID NO:5, and is used to screen humancells for aberrant, including no detectable expression, of the salvadornucleic acid.

As used herein, a “disorder characterized by aberrant expression of asalvador molecule” refers to a disorder in which there is a detectabledifference in the expression levels of salvador molecule(s) and/orMutant salvador molecule(s) in selected cells of a subject compared tothe control levels of these molecules in the cells obtained fromsubjects who do not exhibit tumor growth, metastasis, abnormal cellulardevelopment, abnormal cell proliferation, abnormal cell death, and/orabnormal apoptosis. Thus, a disorder characterized by aberrantexpression of a salvador molecule embraces reduced expression (includingno detectable expression) of a salvador nucleic acid molecule or aSalvador protein compared to control levels of these molecules, as wellas enhanced expression of a Mutant salvador nucleic acid molecule orMutant Salvador protein compared to control levels of these molecules.Such differences in expression levels can be determined in accordancewith the diagnostic methods of the invention as disclosed herein.Exemplary disorders that are characterized by aberrant expression of aSalvador molecule include: various cancers, birth defects, andautoimmunity disorders. In general, each of these disorders isassociated with abnormal cell proliferation, abnormal cell death,abnormal cellular growth and abnormal cell maturation/differentiation,and/or abnormal cell-cell interactions which, for example, characterizetumor cell growth and metastasis.

In certain embodiments, the methods of the invention are to diagnose acancer including, but not limited to, kidney cancer, biliary tractcancer, brain cancer (including glioblastomas and medulloblastomas),breast cancer, cervical cancer, choriocarcinoma, colon cancer,endometrial cancer, esophageal cancer, gastric cancer, hematologicalneoplasms, including acute lymphocytic and myelogenous leukemia,multiple myeloma, AIDS associated leukemias and adult T-cell leukemialymphoma, intraepithelial neoplasms, including Bowen's disease andPaget's disease, liver cancer, lung cancer, lymphomas, includingHodgkin's disease and lymphocytic lymphomas, neuroblastomas, oralcancer, including squamous cell carcinoma, ovarian cancer, includingthose arising from epithelial cells, stromal cells, germ cells andmesenchymal cells, pancreatic cancer, prostate cancer, rectal cancer,renal cancer including adenocarcinoma and Wilms tumor, sarcomas,including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcomaand osteosarcoma, skin cancer, including melanoma, Kaposi's sarcoma,basocellular cancer, squamous cell cancer, testicular cancer, includinggerminal tumors (seminoma, non-seminoma [teratomas, choriocarcinomas]),stromal tumors and germ cell tumors, and thyroid cancer, includingthyroid adenocarcinoma and medullary carcinoma. In the preferredembodiments, the methods of the invention are useful for diagnosingliver cancer, ovarian cancer, testicular cancer, and biliary tractcancer.

In certain embodiments, the methods of the invention are to diagnose abirth defect including, but not limited to, defects resulting fromincreased or decreased cell proliferation, benign and malignantchildhood tumors, neural tube defects, mental retardation, congenitalmalformations resulting from the absence of normal structure, andsupernumerary structures, for example, additional digits.

The method may be used on biological samples including, but not limitedto, amniotic fluid samples, chorionic villus biopsy samples andcell-containing samples from preimplantation embryos, particularlycell-containing samples from early preimplantation embryos stage (cellnumbers equal to or less than 64). Therefore, the invention alsocontemplates prenatal diagnosis of a disorder.

In yet other embodiments, the methods of the invention are useful fordiagnosing an autoimmunity disorder, including, but not limited to,rheumatoid arthritis, multiple sclerosis, type 1 diabetes, psoriasis,and inflammatory bowel disease (IBD).

In the foregoing embodiments, the biological sample can be any nucleicacid- or protein-containing sample obtained from a subject. Exemplarybiological samples are described below. In yet other embodiments, thediagnostic methods are useful for diagnosing the progression of adisorder. According to these embodiments, the methods further involve:detecting in a second biological sample obtained from the subject,expression of a salvador molecule or a Mutant salvador molecule, andcomparing the expression of the salvador molecule or the Mutant salvadormolecule in the first biological sample and the second biologicalsample. In these embodiments, a decrease in the expression of thesalvador molecule in the second biological sample compared to the firstbiological sample or an increase in the expression of the Mutantsalvador molecule in the second biological sample compared to the firstbiological sample indicates progression of the disorder.

In yet other embodiments, the diagnostic methods are useful fordiagnosing the regression of a disorder. According to these embodiments,the methods further involve: detecting in a second biological sampleobtained from the subject, expression of a salvador molecule or a Mutantsalvador molecule, and comparing the expression of the salvador moleculeor the Mutant salvador molecule in the first biological sample and thesecond biological sample. In these embodiments, an increase in theexpression of the salvador molecule in the second biological samplecompared to the first biological sample or a decrease in the expressionof the Mutant salvador molecule in the second biological sample comparedto the first biological sample indicates regression of the disorder.

In certain embodiments, the diagnostic methods of the invention detect asalvador molecule that is a salvador nucleic acid molecule or a Mutantsalvador nucleic acid molecule as described above. In yet otherembodiments, the methods involve detecting a Salvador protein or MutantSalvador protein as described above.

Various detection methods can be used to practice the diagnostic methodsof the invention. For example, the methods can involve contacting thebiological sample with an agent that selectively binds to the salvadormolecule or to the Mutant salvador molecule to detect these molecules.In certain embodiments, the salvador molecule or the Mutant salvadormolecule is a nucleic acid and the method involves using an agent thatselectively binds to the salvador molecule or to the Mutant salvadormolecule, e.g., a nucleic acid that hybridizes to SEQ ID NO:1 or to SEQID NO:3 or to SEQ ID NO:6 under stringent conditions. In yet otherembodiments, the salvador molecule or the Mutant salvador molecule is aprotein and the method involves using an agent that selectively binds tothe Salvador protein or to the Mutant Salvador protein, e.g., a bindingpolypeptide, such as an antibody, that selectively binds to SEQ ID NO:2or to SEQ ID NO:4.

According to still another aspect of the invention, kits for performingthe diagnostic methods of the invention are provided. In general, thekits are nucleic acid-based kits or protein-based kits. According to theformer embodiment, the kits include: one or more nucleic acid moleculesthat hybridize to a salvador nucleic acid molecule (cDNA or genomicsequence) or to a Mutant salvador nucleic acid molecule (cDNA or genomicsequence) under stringent conditions; one or more control agents; andinstructions for the use of the nucleic acid molecules, and agents inthe diagnosis of a disorder associated with aberrant expression of asalvador molecule. Nucleic acid-based kits optionally further include afirst primer and a second primer, wherein the first primer and thesecond primer are constructed and arranged to selectively amplify atleast a portion of an isolated salvador nucleic acid molecule comprisingSEQ ID NO:1 or SEQ ID NO:6. Alternatively, the kits include two isolatednucleic acid molecules, the first consisting of a 20-32 nucleotidecontiguous segment of SEQ ID NO:1 or SEQ ID NO:6 and the secondconsisting of a 20-32 nucleotide contiguous segment of the complement ofSEQ ID NO:1 or SEQ ID NO:6 that does not overlap the first segment.Optionally the isolated nucleic acids are unique fragments of SEQ IDNO:1 or SEQ ID NO:6 or the complements of SEQ ID NO:1 or SEQ ID NO:6.The first and second isolated nucleic acid molecules are designed to actas primers capable of selectively amplifying at least a portion or allof SEQ ID NO:1 or SEQ ID NO:6 and are nonoverlapping to prevent theformation of primer-dimers. One of the primers will hybridize to onestrand of the nucleic acid to be amplified and the second primer willhybridize to the complementary strand of the nucleic acid to beamplified, in an arrangement which permits amplification of the nucleicacid. Selection of appropriate primer pairs is standard in the art. Forexample, the selection can be made with assistance of a computer programdesigned for such a purpose, optionally is followed by testing theprimers for amplification specificity and efficiency.

Alternatively, protein based-kits are provided. Such kits include: oneor more binding polypeptides that selectively bind to a Salvador proteinor to a Mutant Salvador protein; one or more control agents; andinstructions for the use of the binding polypeptides, and agents in thediagnosis of a disorder associated with aberrant expression of asalvador molecule. In the preferred embodiments, the bindingpolypeptides are antibodies or antigen-binding fragments thereof, suchas those described above. In these and other embodiments, certain of thebinding polypeptides bind to the Mutant Salvador protein but do not bindto the Salvador protein to further distinguish the expression of theseproteins in a biological sample.

As used herein, the term “subject” is used to describe a human,non-human primate, cow, horse, pig, sheep, goat, dog, cat, rodent,insect such as Drosophila, or nematode such as C. elegans. In allembodiments human and Drosophila salvador molecules and human subjectsare preferred.

The biological sample can be located in vivo or in vitro. For example,the biological sample can be a tissue in vivo and the agent specific forthe tumor associated nucleic acid molecule or polypeptide can be used todetect the presence of such molecules in kidney tissue (e.g., forimaging portions of the tissue that express the tumor associated geneproducts). Alternatively, the biological sample can be located in vitro(e.g., a blood sample, tumor biopsy, tissue extract). In a particularlypreferred embodiment, the biological sample can be a cell-containingsample, more preferably a sample containing tumor cells. Samples oftissue and/or cells for use in the various methods described herein canbe obtained through standard methods. Samples can be surgical samples ofany type of tissue or body fluid. Samples can be used directly orprocessed to facilitate analysis (e.g., by parafin embedding). Exemplarysamples include a cell, a cell scraping, a cell extract, a blood sample,a tissue biopsy, including punch biopsy, a tumor biopsy, cells from apreimplantation embryo, a bodily fluid, a tissue, or a tissue extract orother methods.

The invention also provides treatment methods. As used herein,“treatment” includes preventing, delaying, abating or arresting theclinical symptoms of a disorder characterized by aberrant expression ofa salvador molecule. Thus, treatment includes reducing or preventingtumor cell growth, proliferation, and/or metastasis, as well as reducingor preventing any manifestation of abnormal cellmaturation/differentiation, cell growth, proliferation, or death(including apoptosis).

In general, the treatment methods involve administering an agent toincrease expression of a salvador molecule and/or reduce expression of aMutant salvador molecule. Thus, these methods include gene therapyapplications. In certain embodiments, the method for treating a subjectwith a disorder characterized by aberrant expression of a salvadormolecule, involves administering to the subject an effective amount of asalvador nucleic acid molecule to treat the disorder. An exemplarymolecule for inhibiting expression of a Mutant salvador nucleic acidmolecule is an anti-sense molecule that is selective for the mutantnucleic acid and that does not inhibit expression of the salvadornucleic acid molecule. Alternatively, the method for treating a subjectwith a disorder characterized by aberrant expression of a Salvadormolecule involves administering to the subject an effective amount of aSalvador protein to treat the disorder. In yet another embodiment, thetreatment method involves administering to the subject an effectiveamount of a binding polypeptide to inhibit a Mutant Salvador proteinand, thereby, treat the disorder. In certain preferred embodiments, thebinding polypeptide is an antibody or an antigen-binding fragmentthereof; more preferably, the antibodies or antigen-binding fragmentsare labeled with one or more cytotoxic agents

The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346and in exhibits submitted in the file history of that patent, all ofwhich are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject which contains a defective copy of the gene, and returning thegenetically engineered cell(s) to the subject. Alternatively, afunctional copy of the gene may be introduced into a cell that ismissing a functional copy of the gene. The functional copy of the geneis under operable control of regulatory elements which permit expressionof the gene in the genetically engineered cell(s). Numerous transfectionand transduction techniques as well as appropriate expression vectorsare well known to those of ordinary skill in the art, some of which aredescribed in PCT application WO95/00654. In vivo gene therapy usingvectors such as adenovirus, retroviruses, herpes virus, and targetedliposomes also is contemplated according to the invention.

In preferred embodiments, a virus vector for delivering a nucleic acidmolecule encoding a Salvador protein is selected from the groupconsisting of adenoviruses, adeno-associated viruses, poxvirusesincluding vaccinia viruses and attenuated poxviruses, Semliki Forestvirus, Venezuelan equine encephalitis virus, retroviruses, Sindbisvirus, and Ty virus-like particle. Examples of viruses and virus-likeparticles which have been used to deliver exogenous nucleic acidsinclude: replication-defective adenoviruses (e.g., Xiang et al.,Virology 219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997;Chengalvala et al., Vaccine 15:335-339, 1997), a modified retrovirus(Townsend et al., J. Virol. 71:3365-3374, 1997), a nonreplicatingretrovirus (Irwin et al., J. Virol. 68:5036-5044, 1994), a replicationdefective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA92:3009-3013, 1995), canarypox virus and highly attenuatedvaccinia-virus derivative (Paoletti, Proc. Natl. Acad Sci. USA93:11349-11353, 1996), non-replicative vaccinia virus (Moss, Proc. Natl.Acad. Sci. USA 93:11341-11348, 1996), replicative vaccinia virus (Moss,Dev. Biol. Stand. 82:55-63, 1994), Venzuelan equine encephalitis virus(Davis et al., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev etal., Virology 212:587-594, 1995), and Ty virus-like particle (Allsopp etal., Eur. J. Immunol 26:1951-1959, 1996). In preferred embodiments, thevirus vector is an adenovirus.

Another preferred virus for certain applications is the adeno-associatedvirus, a double-stranded DNA virus. The adeno-associated virus iscapable of infecting a wide range of cell types and species and can beengineered to be replication-deficient. It further has advantages, suchas heat and lipid solvent stability, high transduction frequencies incells of diverse lineages, including hematopoietic cells, and lack ofsuperinfection inhibition thus allowing multiple series oftransductions. The adeno-associated virus can integrate into humancellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression. In addition, wild-type adeno-associated virus infectionshave been followed in tissue culture for greater than 100 passages inthe absence of selective pressure, implying that the adeno-associatedvirus genomic integration is a relatively stable event. Theadeno-associated virus can also function in an extrachromosomal fashion.

In general, other preferred viral vectors are based on non-cytopathiceukaryotic viruses in which non-essential genes have been replaced withthe gene of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Adenoviruses and retroviruses have been approved for human gene therapytrials. In general, the retroviruses are replication-deficient (i.e.,capable of directing synthesis of the desired proteins, but incapable ofmanufacturing an infectious particle). Such genetically alteredretroviral expression vectors have general utility for thehigh-efficiency transduction of genes in vivo. Standard protocols forproducing replication-deficient retroviruses (including the steps ofincorporation of exogenous genetic material into a plasmid, transfectionof a packaging cell line with plasmid, production of recombinantretroviruses by the packaging cell line, collection of viral particlesfrom tissue culture media, and infection of the target cells with viralparticles) are provided in Kriegler, M., “Gene Transfer and Expression,A Laboratory Manual,” W.H. Freeman Co., New York (1990) and Murry, E. J.Ed. “Methods in Molecular Biology,” vol. 7, Humana Press, Inc.,Cliffton, N.J. (1991).

Preferably, the foregoing nucleic acid delivery vectors: (1) containexogenous genetic material that can be transcribed and translated in amammalian cell and that can suppress tumor cell growth and/orproliferation and/or metastasis, and/or other abnormal cellmaturation/differentiation, cell growth, cell proliferation, cell death,cell migration, and/or cell-cell interaction in a host, and preferably(2) contain on a surface a ligand that selectively binds to a receptoron the surface of a target cell, such as a mammalian cell, and therebygains entry to the target cell.

Various techniques may be employed for introducing nucleic acidmolecules of the invention into cells, depending on whether the nucleicacid molecules are introduced in vitro or in vivo in a host. Suchtechniques include transfection of nucleic acid molecule-CaPO₄precipitates, transfection of nucleic acid molecules associated withDEAE, transfection or infection with the foregoing viruses including thenucleic acid molecule of interest, liposome mediated transfection, andthe like. For certain uses, it is preferred to target the nucleic acidmolecule to particular cells. In such instances, a vehicle used fordelivering a nucleic acid molecule of the invention into a cell (e.g., avirus such as a retrovirus, or a liposome) can have a targeting moleculeattached thereto. For example, a molecule such as an antibody specificfor a surface membrane protein on the target cell or a ligand for areceptor on a target cell can be bound to or incorporated within thenucleic acid molecule delivery vehicle. Especially preferred aremonoclonal antibodies. Where liposomes are employed to deliver thenucleic acid molecules of the invention, proteins which bind to asurface membrane protein associated with endocytosis may be incorporatedinto the liposome formulation for targeting and/or to facilitate uptake.Such proteins include capsid proteins or fragments thereof tropic for aparticular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half life, and the like.Polymeric delivery systems also have been used successfully to delivernucleic acid molecules into cells, as is known by those skilled in theart. Such systems even permit oral delivery of nucleic acid molecules.

The invention provides various research methods and compositions. Thus,according to one aspect of the invention, a method for producing aSalvador protein is provided. The method involves providing a salvadornucleic acid molecule operably linked to a promoter, wherein thesalvador nucleic acid molecule encodes the Salvador protein or afragment thereof; expressing the salvador nucleic acid molecule in anexpression system; and isolating the Salvador protein or a fragmentthereof from the expression system. Preferably, the salvador nucleicacid molecule has SEQ ID NO:1 or SEQ ID NO:3. According to yet anotheraspect of the invention, a method for producing a Mutant Salvadorprotein is provided. This method involves: providing a Mutant salvadornucleic acid molecule operably linked to a promoter, wherein the Mutantsalvador nucleic acid molecule encodes the Mutant Salvador protein or afragment thereof; expressing the Mutant salvador nucleic acid moleculein an expression system; and isolating the Mutant Salvador protein or afragment thereof from the expression system. Preferably, the Mutantsalvador nucleic acid molecule has SEQ ID NO:1 or SEQ ID NO:3 with oneor more deletions, additions, rearrangements, substitutions, ortruncations to encode a Mutant Salvador protein.

The invention further provides efficient methods of identifyingpharmacological agents or lead compounds for agents which mimic thefunctional activity of a salvador molecule. Generally, the screeningmethods involve assaying for compounds which modulate (i.e., up- ordown-regulate) a salvador functional activity. For example, a geneticscreen can be used to identify salvador molecules that function in G1 tofacilitate or inhibit cell cycle entry. This approach stems from ourobservation that the human cyclin-dependent kinase inhibitor p21, whenexpressed in the developing Drosophila eye, can function to arrest cellcycle progression of precursor cells and results in the development of arough eye due to a deficit of retinal cells. This rough eye phenotype isextremely sensitive to levels of endogenous G1 regulators and interactswith mutations in DmcycE (cyclin E), dE2, and roughex, but not withmutations in cell cycle regulators that function at other stages of thecell cycle. Thus, flies overexpressing p21 can be used to identifyadditional salvador molecules and Mutant salvador molecules thatregulate entry into or exit from the cell cycle. (See, e.g., theExamples).

A wide variety of assays for pharmacological agents can be used inaccordance with this aspect of the invention, including, labeled invitro protein-protein binding assays, electrophoretic mobility shiftassays, immunoassays, cell-based assays such as two- or three-hybridscreens, expression assays, apoptosis assays, etc. An assay mixturecomprises a candidate pharmacological agent. Typically, a plurality ofassay mixtures are run in parallel with different agent concentrationsto obtain a different response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e., at zeroconcentration of agent or at a concentration of agent below the limitsof assay detection. Candidate agents encompass numerous chemicalclasses, although typically they are organic compounds. Preferably, thecandidate pharmacological agents are small organic compounds, i.e.,those having a molecular weight of more than 50 and less than about2500, preferably less than about 1000 and, more preferably, less thanabout 500. Candidate agents comprise functional chemical groupsnecessary for structural interactions with proteins and/or nucleic acidmolecules, and typically include at least an amine, carbonyl, hydroxylor carboxyl group, preferably at least two of the functional chemicalgroups and more preferably at least three of the functional chemicalgroups. The candidate agents can comprise cyclic carbon or heterocyclicstructure and/or aromatic or polyaromatic structures substituted withone or more of the above-identified functional groups. Candidate agentsalso can be biomolecules such as peptides, saccharides, fatty acids,sterols, isoprenoids, purines, pyrimidines, derivatives or structuralanalogs of the above, or combinations thereof and the like. Where theagent is a nucleic acid molecule, the agent typically is a DNA or RNAmolecule, although modified nucleic acid molecules as defined herein arealso contemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs of the agents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc., which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease is inhibitors, antimicrobial agents,and the like may also be used.

An exemplary binding assay is described herein. In general the mixtureof the foregoing assay materials is incubated under conditions whereby,but for the presence of the candidate pharmacological agent, thesalvador molecule or the Mutant salvador molecule specifically binds thebinding agent (e.g., antibody, complementary nucleic acid). The order ofaddition of components, incubation temperature, time of incubation, andother parameters of the assay may be readily determined. Suchexperimentation merely involves optimization of the assay parameters,not the fundamental composition of the assay. Incubation temperaturestypically are between 4° C. and 40° C. Incubation times preferably areminimized to facilitate rapid, high throughput screening, and typicallyare between 0.1 and 10 hours.

After incubation, the presence or absence of specific binding betweenthe salvador molecule or the Mutant salvador molecule and one or morebinding agents is detected by any convenient method available to theuser. For cell free binding type assays, a separation step is often usedto separate bound from unbound components. The separation step may beaccomplished in a variety of ways. Conveniently, at least one of thecomponents is immobilized on a solid substrate, from which the unboundcomponents may be easily separated. The solid substrate can be made of awide variety of materials and in a wide variety of shapes, e.g.,microtiter plate, microbead, dipstick, resin particle, etc. Thesubstrate preferably is chosen to maximum signal to noise ratios,primarily to minimize background binding, as well as for ease ofseparation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromotograpic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, non-specific protein, etc. When the solid substrateis a magnetic bead, the beads may be washed one or more times with awashing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as two- or three-hybrid screens. For cell free binding assays, oneof the components usually comprises, or is coupled to, a detectablelabel. A wide variety of labels can be used, such as those that providedirect detection (e.g., radioactivity, luminescence, optical or electrondensity, etc.) or indirect detection (e.g., epitope tag such as the FLAGepitope, enzyme tag such as horseradish peroxidase, etc.). The label maybe bound to a salvador binding partner (e.g., polypeptide), orincorporated into the structure of the binding partner.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions,nonradioactive energy transfers, etc., or indirectly detected withantibody conjugates, strepavidin-biotin conjugates, etc. Methods fordetecting the labels are well known in the art.

According to yet another aspect of the invention, a method foridentifying a salvador molecule or a Mutant salvador molecule isprovided. In certain embodiments, the putative salvador molecule isderived from a human cell, a Drosophila, or a nematode (e.g., C.elegans). The method for identifying a salvador molecule or a Mutantsalvador molecule involves: (a) introducing a putative salvador moleculeor a Mutant salvador molecule into a cell; and (b) detecting a salvadorfunctional activity. The salvador functional activity is selected fromthe group of activities consisting of binding to a cognate molecule(e.g., a warts/LATS molecule) containing a PPPY motif which binds to theWW domain of a Salvador protein, modulating cellmaturation/differentiation, modulating cell growth, modulating cellproliferation, and modulating cell death.

Warts/LATS was originally identified in Drosophila and refers to thesame gene having different names assigned by different laboratories.LATS has GenBank accession number U29608 (SEQ ID NO: 18) for theDrosophila melanogaster large tumor suppressor (lats) mRNA, longtranscript, complete cds; warts has GenBank accession number L39837 (SEQID NO: 19) for the Drosophila melanogaster tumor suppressor (warts) mRNAexons 1-8, complete cds. The PPPY motif in the warts LATS protein is atposition 541-544 of SEQ ID NO: 18 and SEQ ID NO: 20. As used herein, awarts/LATS molecule refers to a nucleic acid or protein molecule thatcomprises a portion of warts/LATS molecule containing the PPPY motif (orwhich encodes this motif). Preferably, the warts/LATS molecule containsa sufficient number of amino acids on either side of the PPPY motif toretain sufficient secondary and, optionally, tertiary structure in themolecule to allow binding of the PPPY motif to a Salvador protein. Ingeneral, the warts/LATS molecule contains at least from about 1 to about50 amino acids (and every integer therebetween) from the naturalwarts/LATS protein on either or both of the 5′ and 3′ terminal ends ofthe PPPY motif. The invention also provides nucleic acids which encodethese portions of the naturally occurring warts/LATS molecule. Thus,these portions of the naturally occurring warts/LATS molecules retaintheir ability to selectively bind to a Salvador protein and can be used,for example, in binding assays to identify agents which modulate thisinteraction and to further define the pathways involving thisinteraction.

The human warts/LATS homologs, LATS1 and LATS2, have GenBank accessionnumbers AF104413 (SEQ ID NO: 21) and AB028019 (SEQ ID NO:23),respectively; although the cDNA sequence for LATS2 is incomplete. Likethe Drosophila warts/LATS gene, the human homolog contains a PPPY motifwhich is believed to bind to a WW domain in the human Salvador protein.

According to still another aspect of the invention, a method foridentifying a salvador modulating agent that modulates a salvadormolecule-cognate interaction is provided. As used herein, a “cognate” isa molecule to which a salvador molecule binds. The method involves: (a)contacting a salvador molecule with a cognate, in the presence of aputative modulating agent, under conditions to allow the salvadormolecule to bind to the cognate (e.g., warts/LATS); and (b) detectingsalvador molecule binding to the cognate. A change in salvador moleculebinding to the cognate in the presence of the putative modulating agentcompared to salvador molecule binding to the cognate in the absence ofthe cognate indicates that the agent is a salvador modulating agent,that is, a molecule that affects the binding properties of a salvadormolecule and a cognate. In general, detecting comprises detecting achange in a parameter selected from salvador molecule binding to itscognate, cell maturation/differentiation, cell growth, cellproliferation, and/or cell death.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example 1 Introduction to Identification of Salvador Molecules

Mutations in the salvador gene in Drosophila were identified bydetecting mutant cells having a proliferative or growth advantage overwild type cells using FLP-induced mitotic recombination in the eye(described in detail below and in Tapon, N., et al., Cell, 110(4):467,2002, hereby incorporated by reference).

More than 200 mutations were found using the above-identified assay.These mutations include the Mutant salvador nucleic acid moleculeshaving SEQ ID NOs: 15, 17, and 19; and their gene products having SEQ IDNOs: 16, 18, and 20.

Clones of the mutant Drosophila cells were generated in wing and notumtissues and resulted in the appearance of tumor-like growths on thistissues. Further characterization of the clones included visualizingcell proliferation using BrdU incorporation and TUNEL labeling tovisualize cell death. The results of these studies indicated that themutant Drosophila cells do not arrest in G1 phase and continue toS-phase and that there is almost no apoptosis in mutant clones.

Meiotic mapping of the Drosophila salvador gene identified CG13832 asthe genomic sequence which includes an open reading frame correspondingto the cDNA for the Drosophila salvador gene having SEQ ID NO:3 and itsprotein product having SEQ ID NO:4. Mutant Drosophila salvador nucleicacid molecules were sequenced in accordance with standard procedures.The Mutant Drosophila salvador molecules of the invention that have beenidentified in accordance with this procedure include Mutant salvadornucleic acid molecules (SEQ ID NOs:15, 17, and 19) and Mutant Salvadorproteins (SEQ ID NOs:16, 18, and 20).

The human homologue to the Drosophila salvador gene was identified asthe hWW45 gene (GenBank accession number NM0218118) reported by Valverde(Biochem. Biophys. Res. Commun. 276 990-998, 2000), with the humansalvador cDNA having SEQ ID NO:1 and the encoded human Salvador proteinhaving SEQ ID NO:2.

Screening for the salvador gene in human cancer cells lines wasaccomplished using a probe (SEQ ID NO:5) for the human salvador gene, inaccordance with the procedures described below.

The results obtained using SEQ ID NO:5 as the hybridization probeestablished that all coding sequences of the human salvador gene aredeleted in the ACHN human kidney tumor cell lines and exons 3, 4, and 5(nucleotides 25027-25297, nucleotides 27622-27765, and nucleotides33131-34873, of Seq ID NO:6, respectively) are deleted in the 786-0human kidney tumor cell line.

Mutant human salvador molecules are identified in the accordance withthe above-described procedures and are sequenced in accordance withstandard procedures. In general, the Mutant salvador nucleic acidmolecules contain a sequence which is the same as SEQ ID NO:1 or SEQ IDNO:3, with the exception that the sequence includes one or moremutations, e.g., point mutations, deletion mutations, or truncations,such that the Mutant salvador nucleic acid molecule does not encode afunctional Salvador protein. Rather, the Mutant salvador nucleic acidmolecules encode a Mutant Salvador protein, i.e., a protein which doesnot exhibit Salvador protein functional activity. In certain preferredembodiments, the Mutant human salvador nucleic acid molecules aretruncated forms of SEQ ID NO: 1 which lack one or more WW domains. TheWW domains are located at positions 813-911 and 918-1016 of SEQ ID NO:1.In related embodiments, the Mutant human Salvador proteins are truncatedforms of SEQ ID NO:2 which lack one or more WW domains. The WW domainsin the protein sequence are located at positions 200-232 and 235-267 ofSEQ ID NO:2.

Example 2 Screening Methods and Results

A genetic screen in Drosophila was used to identify genes that restrictgrowth and proliferation. Once the Drosophila salvador gene wasidentified, database searches were performed to identify the humanhomologue. Once identified, tumor cell lines were examined for mutationsin the putative tumor suppressor gene.

In order to identify genes that restrict cell numbers in vivo, a geneticscreen in Drosophila was conducted to identify mutations in genes thatresult in mutant cells having even a subtle growth or proliferativeadvantage over their wild-type neighbors. Using FLP-induced mitoticrecombination in the eye, clones of mutant tissue were generated andcompared in size to the wild-type twin spots generated from the samerecombination event. After screening the four major autosomal arms whichtogether account for approximately 80% of the genome, more than 200mutations that result in this phenotype were identified. Of these, 23loci are represented by more than a single allele. These includemutations in Drosophila homologues of known human tumor suppressor genesincluding PTEN (5 alleles) and the Tuberous Sclerosis Complex (Tsc1, 3alleles; Tsc2, 2 alleles).

Another locus identified in the screen, represented by 3 alleles is anovel gene salvador which corresponds to the transcription unitdesignated CG13832. Mutations in salvador result in inappropriate cellproliferation and a failure of cell cycle exit. Moreover, mutant cellsappear to be resistant to some of the apoptotic signals that result inthe death of is their wild-type neighbors. Clones of mutant salvadorcells can give rise to tumorous outgrowths. We have cloned the salvadorgene. It encodes a novel protein with two WW-domains with highlyconserved orthologues in C. elegans and mammals. The human homologue ofsalvador, hWW45 (accession number NM021818), containing two WW-domainsfrom amino acids 200 to 232 and 235 to 267, has been identifiedpreviously but its function was not known. We have now shown that hWW45appears to be deleted or rearranged in at least two human kidney cancercell lines.

To identify genes that restrict cell growth or cell numbers in vivo, ascreen in the Drosophila eye for mutations that increased the relativerepresentation of mutant tissue compared to wild-type tissue wasconducted (Tapon, N., et al. (2001) Cell 105, 345-355). UsingFLP/FRT-induced mitotic recombination, clones of white mutant tissuewere compared in size to sister clones of red wild-type tissue. Thoseflies whose eyes contained an excess of mutant over wild-type tissuewere retained. Mutations in at least 23 distinct loci that elicit thisphenotype were identified. These included negative regulators of cellproliferation such as archipelago (ago) as well as homologs of humantumor-suppressor genes including PTEN, TSC1, and TSC2 (Moberg, K. H., etal. (2001) Nature 413, 311-316; Tapon, N., et al. (2001) Cell 105,345-355).

Three alleles of salvador were identified using this screen. A fourthallele, salvador⁴, was isolated by Jessica Treisman and kindly provided.Salvador¹ and salvador² generated eyes that have an increasedrepresentation of mutant tissue over wild-type tissue when compared tothe parent chromosome. Salvador³ elicited a more severe phenotype; inaddition to a further increase in the representation of mutant tissue,the mutant tissue protruded from the eye in folds. Salvador⁴ exhibitedan intermediate phenotype. Clones of salvador³ mutant s tissue generatedin other parts of the fly including the notum and haltere also displayedoutgrowths. All four alleles were lethal when homozygous, in trans toeach other or in trans to the deletion Df(3R)hh that spans the salvadorlocus.

In salvador¹ clones in the adult retina, almost all the ommatidiacontained the normal complement of eight photoreceptor cells. However,there was increased spacing between adjacent ommatidia. In contrast towild-type retinas from late pupae that contained a single layer ofinterommatidial cells, mutant clones contained many additionalinterommatidial cells. Generation of salvador¹ mutant clones in a white⁺background indicated that most of these additional interommatidial cellscontained pigment. Thus, these cells underwent terminal differentiation.The more disorganized retinas of the salvador³ allele displayed all ofthese phenotypic abnormalities. In addition, almost half of theommatidia in salvador³ clones lacked one or more photoreceptor cells.

Example 3 Salvador Promotes Cell Cycle Exit

In wild-type imaginal discs, S phases, as visualized by BrdUincorporation, were observed anterior to the morphogenetic furrow (MF)and as a single stripe of incorporation posterior to the furrow referredto as the second mitotic wave (SMW). In salvador clones, manyBrdU-incorporating nuclei were observed posterior to the SMW. Clonesspanning the MF had some BrdU-incorporating nuclei in the anterior halfof the MF, a region that is normally composed of cells arrested in G1.Using the anti-phosphohistone H3 antibody, additional cells in mitosiswere also visualized in salvador mutant clones posterior to the MF,suggesting that at least some of these cells had completed additionalcell cycles. BrdU incorporation persisted in mutant clones during thefirst 12 hr after puparium formation (APF) but ceased by 24 hr APF.Thus, salvador mutant cells continued to proliferate for 12 to 24 hrafter wild-type cells stopped dividing but eventually were able to exitfrom the cell cycle to undergo terminal differentiation.

In cycling cells in the anterior portion of the eye imaginal disc, thedistribution of mutant cells in the cell cycle, as assessed by flowcytometry, was extremely similar to that of wild-type cells. The mutantcells were very slightly smaller than their wild-type counterparts.Posterior to the MF, mutant populations had an increased proportion ofcells in S and G2, indicating that mutant cells continued to cycle inthis portion of the disc. Mutant cells were of normal size. Thepopulation doubling times of clones of mutant cells and wild-type cellsgenerated in the wing imaginal disc during the proliferative phase ofdevelopment did not differ significantly. Thus, when proliferating,mutant cells behaved like wild-type cells. However, exit from the cellcycle was delayed in salvador cells.

Elevated levels of Cyclin E protein were found in the basal nuclei ofsalvador clones posterior to the MF. These were the nuclei of theundifferentiated cells that continued to proliferate in salvador clones.They were examined for levels of cyclin E RNA. When salvador clones weregenerated using eyFLP (Newsome, T. P., et al. (2000) Development 127,851-860), a large proportion of cells in third instar discs were mutant,and these discs contained large patches of mutant tissue.

In wild-type discs, cyclin E RNA was expressed in a narrow stripeimmediately posterior to the morphogenetic furrow. In discs containingsalvador clones, the stripe of expression was broader and more intense,indicating that cyclin E RNA levels were elevated in these discs. Thus,the increased level of Cyclin E protein is likely to have resulted from,at least in part, from an increase in cyclin E RNA levels.

Example 4 Salvador is Required for Apoptosis in the Eye Imaginal Disc

In wild-type eyes, excessive interommatidial cells are eliminated by awave of apoptosis that is evident in 38 hr pupal retinas (Wolff, T. andReady, D. F. (1993) Pattern formation in the Drosophila retina. In TheDevelopment of Drosophila melanogaster, M. Bate, and A. Martinez Arias,eds. (Plainview, New York: Cold Spring Harbor Laboratory Press)1277-1325). Even in salvador mutant clones, cell proliferation, asassessed by BrdU incorporation, had ceased within 24 hr APF. When mosaicretinas were examined 38 hr APF, cell death was mostly confined to thewild-type portions of the retina. Thus, the apoptotic cell deaths thatare part of normal retinal development appear to require salvadorfunction.

Apoptosis in the pupal retina requires hid function, since hid mutantsdisplay additional interommatidial cells (Kurada, P. and White K. (1998)Cell 95, 319-329). Hid is thought to induce caspase activation bybinding to the DIAP1 protein and preventing it from inhibiting caspasefunction (Goyal, L., et al. (2000) EMBO J. 19, 589-597; Lisi, S., et al.(2000) Genetics 154, 669-678; Wang, S. L. (1999) Cell 98, 453463).Overexpression of hid using the eye-specific GMR promoter generated asmall eye (Hay, B. A., et al. (1995) Cell 83, 1253-1262). The inductionof cell death by hid was severely impaired in salvador mutant clones. Asa consequence, eyes derived from GMR-hid-expressing discs that containsalvador mutant clones were larger than those derived from wild-typediscs that express GMR-hid. Since salvador function is required forhid-induced cell death, salvador is believed to function eitherdownstream of hid or in a parallel pathway.

Several recent studies have shown that another mechanism by which Hidand Rpr activate caspases is by inducing the autoubiquitination of DIAP1and targeting it for degradation by the proteasome (Hays, R., et al.(2002) Nat. Cell Biol. 4, 425431; Holley, C. L., et al. (2002) Nat. CellBiol, 4, 439444; Ryoo, H. D., et al. (2002) Nat. Cell Biol. 4, 432-438;Wilson, R., et al. (2002) Nat. Cell Biol. 4, 445-450; Wing, J. P., etal. (2002) Nat. Cell Biol. 4, 451456; and Yoo, S. J., et al. (2002) Nat.Cell Biol. 4, 416424). It was observed that DIAP1 levels were markedlyelevated in salvador clones in the larval eye disc, and remainedelevated in the interommatidial cells in mutant clones in the pupal eyedisc, where a reduction of apoptosis was observed. Thus, it is believedthat increased levels of DIAP1 in salvador cells may be able to overcomethe effect of many proapoptotic signals.

To examine DIAP1 RNA levels, in situ hybridization was used to examine20 wild-type discs and 20 mutant discs. The presence of salvador (GFP⁻)clones in the mutant discs was confirmed by examining the discs byfluorescence microscopy prior to hybridization. There was a modest levelof DIAP1 RNA expression posterior to the furrow in both populations ofdiscs and no evidence of increased DIAP1 RNA in the discs containingsalvador clones. Thus, it is believed that this level of detection, theincreased DIAP1 expression in salvador cells does not result fromincreased transcription.

In wild-type eye discs, DIAP1 protein was expressed at higher levelsposterior to the morphogenetic furrow. DIAP1 protein levels weredownregulated by GMR-rpr or, to a lesser extent, by GMR-hid expression.In salvador mutant clones expressing GMR-rpr, DIAP1 protein levelsremained elevated. Similar results were observed with GMR-hid. Thus, itis believed that neither GMR-rpr nor GMR-hid is capable ofdownregulating the elevated levels of DIAP1 sufficiently in salvadorclones to activate caspases.

Expression of hid or reaper (rpr) in the eye imaginal disc resulted inactivation of the effector caspase Drice. An antibody that recognizesthe cleaved (activated) form of Drice (Yoo, S. J., et al. (2002) Nat.Cell Biol. 4, 416-424) was used to stain eye discs expressing GMR-hid orGMR-rpr. In wild-type cells, Drice was activated by GMR-hid or GMR-rpr.However, in clones of salvador tissue, Drice activation by eitherGMR-hid or GMR-rpr was almost completely blocked. To counteract thepossibility of convolutions in the disc, stainings which wereprojections of confocal Z series (12 individual frames) spanning theentire thickness of the eye disc, excluding the peripodial membranes,were analyzed. At least 30 discs per genotype from three independentexperiments were carefully examined to confirm the results. Theseexperiments indicated that Salvador blocks activation of Drice by bothrpr and hid.

A mutant form of Hid (Hid-Ala5) is resistant to inactivation by MAPkinase phosphorylation (Bergmann, A., et al. (1998) Cell 95, 331-341).GMR-hid-Ala5 was a more potent inducer of cell death, as assessed by theextent of Drice activation in the eye disc, than is GMR-hid. Cell deathinduced by GMR-hid-Ala5 was only partially blocked in salvador clones,indicating that the increased potency of Hid-Ala-5 may have been able toovercome increased DIAP1 levels.

Example 5 Salvador Encodes a Protein with WW Domains and has a HumanOrtholog

The salvador mutations were localized to the interval 93F11-13 to94D10-13 (formerly annotated 94D 4-7 to 94E 1-2). High-resolutionmeiotic mapping localized salvador to a 20 kb region that contained fiveORFs. All five ORFs were sequenced completely and it was found that allfour Salvador chromosomes had truncating mutations in CG13831. The otherfour ORFs did not have any amino acid changes.

Meiotic mapping using multiplying marked chromosomes, and subsequentlyusing deletions, allowed for localization of Salvador to the region 94D4-7 to 94E 1-2 on the cytogenetic map. Salvador was crossed to all thelethal P-element insertions in the region and all of them were able tocomplement Salvador. Thus the cloning of salvador was not facilitated bythe finding of a P-element insertion in the gene. Recombination in maleswas then initiated by the mobilization of a P-element to locate salvadorwith respect to identified P-element insertion. It was determined thatsalvador maps to the right of a P-element in the region of the klingongene and left of a P-element in hedgehog. A recombinant chromosome wasgenerated that had the klingon insertion in cis to salvador. Thechromosome containing the P-element insertion in hedgehog was placed intrans and recombinant chromosomes were identified (by conventionalrecombination in female meiosis) that lacked both P-elements (white-eyedprogeny). SNPs in the interval were identified that allowed fordistinguishing the parent chromosome used in the screen from thechromosome that had the hedgehog P-element insertion. These SNPs allowedfor identification of the regions where the crossovers had occurred.Using this type of analysis salvador was localized to a region of lessthan 30 kb. At this stage, the Drosophila genomic sequence becameavailable. Two candidate genes in that region included the Drosophilap53 homologue and a transcription unit identified as CG13832. p53sequences from each salvador mutant chromosome were amplified. Nomutations were identified. The CG13832 gene was then sequenced. Each ofthe salvador alleles had a truncating mutation in this ORF.

Five independent cDNA clones of CG13831 were examined by restrictionmapping, and two independent clones were sequenced completely. Thelongest clone was 2.2 kb long, which is in agreement with theapproximate size of the RNA determined by Northern blotting. Thepredicted ORF, encoding a protein of 608 amino acids, included theentire coding region since there is a stop codon upstream and in-framewith the ATG codon.

The predicted Salvador protein has two WW domains, and its C-terminalportion includes a domain that is likely to adopt the conformation of acoiled-coil. Salvador is most similar to the human protein hWW45(Valverde, P. (2000) Biochem. Biophys. Res. Commun. 276, 990-998) and tothe protein encoded by the C. elegans ORF T10H10.3. WW domains are knownto mediate protein-protein interactions with various proline containingmotifs (Kato, Y., et al. (2001) J. Biol. Chem. 277, 10173-10177).

The more C-terminal WW domain of hWW45 lacks the second conservedtryptophan residue that is required for substrate binding and may not bea functional WW domain. The N-terminal WW domain contains all of theappropriate conserved residues. This putative WW domain is predicted tobelong to the Group I family of WW domains that is predicted to interactwith the PPXY (“PY”) motif.

Although not wishing to be bound by theory, it is believed that salvadorand warts interact via a WW domain-PY motif-dependent interaction andfunction to promote cell cycle exit and apoptosis during development.However, warts may have salvador-independent functions as well. Whilesalvador mutations appeared to result in a subtle increase in growthrate, the very strong overrepresentation of warts mutant tissue in thirdinstar larval discs indicated that warts mutations must cause a muchgreater increase in growth rate.

The CG13832 ORF encodes a 386 amino acid protein which contains two WWdomains and a putative coiled-coil domain. The original annotation ofthe gene in the Gadfly is incomplete and a more recently deposited ESTincludes the entire coding region. The coding region is from nucleotide81 to 1907. The salvador¹ allele changes codon 289 from a Gln (CAG) to astop (TAG). The salvador² changes codon 231 (or 232) from a Gln (CAG) toa stop (TAG) and the salvador³ allele introduces a frame-shift in codon407 by the deletion of a G nucleotide. Thus each mutation results in thesynthesis of a truncated protein that lacks both WW repeats.

hWW45 displays strong sequence similarity to salvador at the amino acidlevel and shows an identical organization of domains. Over theC-terminal 188 amino acids, the two proteins display 47% identity and54% similarity at the amino acid level. The human protein contains twoWW domains from amino acids 200 to 232 and 235-267. This gene has beencalled hWW45 by others but its function was not known. A previousdescription of this human gene (Valverde, P. Biochem. Biophys. Res.Commun. 276 990-998, 2000) contains only sequence information andanalysis of expression in different tissues by Northern blotting butdoes not provide any experimental data that suggest a function.

As described herein, hWW45 was shown to be deleted or rearranged in atleast two human kidney cancer cell lines. It is anticipated that thisgene is either deleted or rearranged in other cells exhibiting abnormalcellular development.

The mutations in salvador¹, salvador², and salvador⁴ result in stopcodons in positions 289, 231, and 160, respectively, that truncate theprotein N-terminal to the WW domains, as shown in FIG. 1. The moreN-terminally salvador⁴ mutation has a more severe phenotype thansalvador¹ or salvador². Surprisingly, the salvador³ mutation, whichelicited the most severe phenotype, maps 3′ to those found in salvador¹and salvador². The salvador³ mutation causes a frameshift and generateda protein consisting of 406 salvador-encoded amino acids derived fromthe use of an alternate open reading frame that has no sequencesimilarity to any protein in the database. Although not wishing to bebound by theory, it is believed that salvador¹, salvador² and salvador⁴proteins may have some residual activity despite the absence of the WWdomains and that salvador³ is a null allele. The salvador³ allele mayhave a more severe phenotype because the novel C-terminal sequences mayhave further impaired its stability or function. Alternatively, thenovel C terminus of the salvador³ protein may have conferred someneomorphic properties. Any such properties, if present, were notapparent in the presence of the wild-type protein, since salvador³/+flies displayed no overt phenotypic abnormalities. In differenttransheterozygous combinations, salvador³ was similar in strength to adeletion. In four independent experiments, salvador¹/salvador³ animalsand salvador¹/Df(3R)EB6 animals had hatching rates of 85.5% (SD 2.5%)and 83.3% (SD 3.2%), respectively (n=40), and 90%-95% of the animals ofeach genotype subsequently failed to grow and died as first instarlarvae. Thus, at least by this criterion, salvador³ behaved like a nullmutation. The abnormalities in cell proliferation and apoptosis wereanalyzed using at least two different salvador alleles and onlyquantitative differences were observed between salvador³ and the weakeralleles.

In the eye disc, salvador was expressed in a stripe in the MF, andexpression decreased in the region of the SMW. Expression increased onceagain posterior to the SMW. Thus, to a first approximation, salvadorexpression coincided with regions of temporary or permanent cell cyclearrest which supports the theory that salvador functions in promotingexit from the cell cycle.

Example 6 Salvador Functions Together with Warts

Clones of cells mutant for warts generate large tumor-like growths inDrosophila (Bryant, P. J., et al. (1993) Devel. Suppl, 239-249; Justice,R. W., et al. (1995) Genes Dev. 9, 534-546; Xu, T., et al. (1995)Development 121, 1053-1063). Its human ortholog LATS1 binds to the cdc2protein kinase in a cell cycle-dependent manner and inhibits itsactivity (Tao, W., et al. (1999) Nat. Genet. 21, 177-181). Thus, it hasbeen suggested that excessive Cyclin A/cdc2 may cause excessive cellproliferation by promoting both the G1/S and G2/M transitions. Theinteraction between warts and cdc2, however, does not explain theexcessive and inappropriate growth (mass accumulation) that appears todrive the cell proliferation in clones of warts mutant cells. The defectin cell death in warts cells is also not easily accounted for by theinteraction of Warts with cdc2.

One candidate for a Salvador-interacting protein is encoded by the warts(wts; also known as LATS) gene (Bryant, P. J., et al. (1993) Devel.Suppl, 239-2495; Justice, R. W., et al. (1995) Genes Dev. 9, 534-546;Xu, T., et al. (1995) Development 121, 1053-1063) that encodes aserine-threonine kinase. Clones of warts tissue generated outgrowthsthat resembled tumors. Nine alleles of warts were identified in ourscreen, and the phenotype of salvador³ was similar to that elicited byhypomorphic mutations in warts. Null alleles of warts displayed a moresevere phenotype. Like salvador, warts clones in the pupal retina hadadditional interommatidial cells. Larval imaginal discs containing largewarts clones are enlarged and convoluted (Justice, R. W., et al. (1995)Genes Dev. 9, 534-546; Xu, T., et al. (1995) Development 121,1053-1063). Larval eye discs that contain eyFLP-induced warts cloneswere composed mostly of mutant tissue with small regions of wild-typetissue. Many additional BrdU-incorporating nuclei were observed inmutant clones posterior to the SMW. As observed with salvador, thestripe of cyclin E RNA expression was also broadened in these discs.Moreover, the normal cell death that occurs in the pupal retina wasalmost completely abolished in warts mutant clones. Thus, as forsalvador, warts mutations generate additional interommatidial cellsresulting from both increased cell proliferation posterior to the SMW aswell as reduced apoptosis in the pupal retina. In addition, Driceactivation induced by GMR-hid is markedly diminished in warts clones.

Overexpression of salvador alone using the GMR promoter (Hay, B. A., etal. (1995) Cell 83, 1253-1262) had no effect, and overexpression ofwarts generated subtle irregularities in ommatidial architecture.However, combined overexpression of salvador and warts resulted in asmaller eye where the ommatidial pattern is highly irregular. Thiseffect appeared to reflect a synergistic increase in cell death in theeye discs of flies that express both transgenes as well as a minoreffect on reducing cell proliferation associated with the SMW.

Thus, although not wishing to be bound by theory, it is believed thatsalvador and warts may function in the same pathway and bind to eachother. Indeed, the Salvador protein has a Group I WW domain that ispredicted to interact with the PPXY (PY) motif, five of which are foundin the Warts protein. To test whether Drosophila Salvador and Wartsproteins could physically interact, a GST pull-down assay was employed.The region containing the two potential WW domains of Salvador was fusedto GST and incubated with cell lysates that expressed Myc-tagged Wartsprotein. Using this assay, Warts was found to interact specifically withthe region of Salvador that contained the WW domain. Furthermore, a 15amino acid peptide, designed to mimic one of the PY motifs of Warts, wasfound to inhibit the interaction between the WW domain region ofSalvador and Warts. An identical peptide where the tyrosine residue thatis required for interaction with type I WW domains had been replaced byan alanine did not prevent this interaction. Thus, at least under theconditions of this experiment, Salvador and Warts interact in a WWdomain- and PY motif-dependent fashion. It is believed that an analogousinteraction occurs in vivo.

Discs containing clones of the warts null allele, warts^(latX1) (Xu, T.,et al. (1995) Development 121, 1053-1063), are much larger than discscontaining salvador³ clones. If all salvador functions were wartsdependent, the double mutant phenotype would not be more severe than thewarts phenotype. When mutant clones were generated with eyFLP, averagedisc sizes were 39,669 pixels (SD 10,401) for salvador³, warts^(latsX1)double mutant discs and 31,360 pixels (SD 5260) for warts^(latsX1) discs(n=20). Thus, the double mutant discs were significantly larger than thewarts^(latsX1) discs (p<0.01). Thus, while salvador and warts appear tofunction together in certain ways, they are believed to have functionsthat are independent of each other as well.

Example 7 The Human Ortholog of Salvador, hWW45, is Mutated in CancerCell Lines

Since mutations in salvador lead to excessive cell proliferation andreduced cell death, hWW45 was tested to see if it is a mutational targetin cancer. hWW45 maps to the chromosomal region 14q13-14q23 (Valverde,P. (2000) Biochem. Biophys. Res. Commun. 276, 990-998), a locus that issubject to allelic loss in a variety of cancers, including renalcancers, ovarian cancers, and malignant mesothelioma. The entire codingregion of hWW45 in a panel of 52 tumor-derived cell lines representing abroad range of tissue types was sequenced. One colon cancer cell line,HCT15, had a heterozygous C to A mutation at nucleotide 554, resultingin a substitution of aspartic acid for alanine at codon 185. Thismutation was not present in 185 population-based controls (370chromosomes), indicating that it is not a common polymorphism. HCT15carries a mutation in the mismatch repair gene MSH6, which appears toenhance the frequency of point mutations in other genes. Moresignificantly, two renal cancer cell lines, ACHN and 786-O, were foundto have deletions involving hWW45. The normal allele was not present ineither cell line, indicating that these cell lines are either homozygousor hemizygous for the deletion. The hWW45 transcript was undetectable byRT-PCR in both cell lines, and a Southern blot using a probe derivedfrom the 3′ portion of the gene demonstrated that this part of the genewas absent in both cell lines. In cell line 786-O, PCR analysis ofgenomic DNA indicated that there is a deletion of ˜157 kb with the 5′breakpoint between exons 2 and 3 of hWW45. The deletion in ACHN of ˜138kb encompassed the entire gene. The common region of overlap betweenthese two deletions is only 21 kb, containing exons 3-5 of hWW45. Noother transcription units were identified within this 21 kb interval,using the GENSCAN exon prediction program. Thus, deletions thatinactivate the human ortholog of salvador were identified in at leasttwo cancer cell lines.

Example 8 Role of Salvador in Promoting Cell Cycle Exit

In the eye disc, salvador clones contain cells that continued toproliferate for 12-24 hr after their normal counterparts stop dividing.Studies of cycling cells showed almost no differences between wild-typeand mutant populations. However, given that mutant clones contain moreommatidia than wild-type twin spots, accelerated growth must haveoccurred in mutant tissue anterior to the furrow. Even a relativelyminor growth advantage exhibited by mutant cells at every cell cycle caneventually result in increased clone size when amplified by theapproximately nine rounds of cell division that occur in the eyeprimordium prior to the passage of the MF. A subtle change in cell cycleparameters may not be easily detectable.

In salvador clones, elevated Cyclin E protein levels were observed inthe basal nuclei posterior to the MF in the eye imaginal disc. Thesecells normally stop dividing when they downregulate Cyclin E proteinlevels. In discs containing many salvador clones, the stripe of cyclin ERNA expression is broader and more intense. Thus, the increased level ofCyclin E protein was, at least in part, a result of elevated cyclin ERNA levels. Thus, an inability to downregulate Cyclin E/cdk activity maybe the result of increased levels of cyclin E RNA as occurs in salvadorclones, impaired protein degradation (Moberg, K. H., et al. (2001)Nature 413, 311-316), or reduced levels of the cdk inhibitor Dacapo (deNooij, J. C., et al. (1996) Cell 87, 1237-1247; Lane, M. E., et al.(1996) Cell 87, 1225-1235). In each case, cell cycle exit is delayed.

Example 9 Role of Salvador in Regulating Cell Death

Elevated DIAP1 levels were likely to underlie the absence of thedevelopmentally regulated apoptosis in salvador clones in the pupalretina as well as the resistance to hid-induced and rpr-inducedapoptosis in the larval imaginal disc. The elevated DIAP1 levelsappeared to result from alterations in posttranscriptional regulation ofDIAP1 expression. Recent work has shown that both Rpr and Hid candownregulate DIAP1 levels either by promoting the autoubiquitination ofDIAP1 or by causing a generalized inhibition of translation thatespecially impacts proteins with a short half-life such as DIAP1 (Hays,R., et al. (2002) Nat. Cell Biol. 4, 425-431; Holley, C. L., et al.(2002) Nat. Cell Biol. 4, 439-444; Ryoo, H. D., et al. (2002) Nat. CellBiol. 4, 432-438; Wilson, R., et al. (2002) Nat. Cell Biol. 4, 445-450;Wing, J. P., et al. (2002) Nat. Cell Biol. 4, 451-456; Yoo, S. J., etal. (2002) Nat. Cell Biol. 4, 416-424). Either of these mechanisms islikely to be less efficient in cells that already have elevated levelsof DIAP1.

The findings described herein indicate that Salvador normally functionsto downregulate the basal level of DIAP1 protein. In the absence ofSalvador, higher levels of DIAP1 accumulate. This increases the level ofHid or Rpr activity that is required to overcome DIAP1-mediatedinhibition of caspase activation. Consistent with this model, the morepotent form of Hid, Hid-Ala5, is able to partially overcome theincreased levels of DIAP1 in salvador clones and induce a low level ofcaspase activity.

It is believed that Salvador is capable of regulating both cell cycleexit and apoptosis by virtue of its ability to modulate the levels oftwo key regulators—Cyclin E and DIAP1. Loss of salvador appears toincrease cyclin E levels transcriptionally and DIAP1 levels by aposttranscriptional mechansim. Since cell number is determined by boththe extent of cell proliferation as well as apoptosis, Salvador couldfunction as a key regulator of cell number by virtue of its ability toregulate both processes.

One of few pathways that can directly regulate both cell proliferationand cell death is the Ras/MAPK pathway. Ras can promote cellproliferation by promoting growth (Prober, D. A. and Egar, B. A. (2000)Cell 100, 435446), and MAP kinase can phosphorylate and inactivate Hidand also reduce Hid transcription (Bergmann, A., et al. (1998) Cell 95,331-341; Kurada, P. and White K. (1998) Cell 95, 319-329). Although notwishing to be bound by theory, the results described herein indicatethat Salvador might function in a distinct pathway. First, no change indiphospho-ERK activity was observed in salvador mutant clones. Second,cell death induced by the MAP kinase-resistant Hid-Ala5 protein (wherefive putative MAPK phosphorylation sites have been mutated to alanines)was also reduced by a loss of salvador function. However, it is stillpossible that Salvador might function downstream of the MAPK familyproteins.

Example 10 Salvador and Warts Orthologs as Tumor Suppressors in Humans

Mice lacking the warts ortholog LATS1 display pituitary hyperplasia anddevelop slow-growing tumors (St. John, M. A., et al. (1999) Nat. Genet.21, 182-186). This contrasts with the dramatic overgowth phenotypeobserved in warts mutants in Drosophila. It is believed that thesedifferences may be due to the presence of other warts homologs (e.g.,LATS2) in mammals that can partially compensate for LATS1 inactivation(St. John, M. A., et al. (1999) Nat. Genet. 21, 182-186).

Although not wishing to be bound by theory, it is believed that thepresence of a single salvador homolog, hWW45, in humans makes it lesslikely that its function is redundant with that of a related gene.Mutations in this gene in three cancer cell lines have been identifiedand it has been shown that two of these cell lines have homozygousdeletions that either disrupt or eliminate the gene. While cell linescan accumulate mutations in culture, these findings neverthelessrepresent a first step in implicating hWW45 in the pathogenesis of humancancer.

Athough chromosomal aberrations have been consistently identified for anumber of human tumors, in most cases the relevant lesion has not beenmolecularly characterized. Many mammalian tumor suppressor genes mustexist that have not yet been identified. The phenotype-based screendescribed herein, which is capable of detecting even subtle increases ingrowth or cell proliferation, has identified a number of genes thatrestrict growth or cell number. For ago and salvador, mutations in theirhuman orrthologs in cancer cell lines have been subsequently identified.Thus, the strategy of conducting phenotype-based screens in modelorganisms followed by a search for mutations in cancer cell lines may beused to identify new tumor suppressor genes.

Example 12 Experimental Procedures

Fly Stocks

w; FRT82B males were mutagenized with ethylmethanesulfonate (EMS), thencrossed either to y w eyFLP; FRT82B P[mini-w, arm-LacZ] or first to w;TM3/TM6B and then individually toy w eyFLP; FRT82B P[mini-w, armLacZ](Tapon, N., et al. (2001) Cell 105, 345-355). Males with mostly whiteeyes were retained and maintained as balanced stocks. Alleles ofsalvador identified were salvador¹, salvador², and salvador³. GMR-hidand 2XGMR-rpr (on the second chromosome) were provided by Kristin White.GMR-hid Ala5 (second chromosome) was provided by Andreas Bergmann.FRT82B LATS^(X1) has been described (Xu, T., et al. (1995) Development121, 1053-1063). Warts^(MGH1), identified in our screen, is a homozygouslethal allele of moderate strength.

Mapping

Salvador mutations failed to complement the lethality of Df(3R)hh, whichdeleted 93F11-13 to 94D10-13. Using P element-mediated malerecombination, the salvador¹ allele was placed in cis to P[lacW]C2-3-33at 94D. The P[lacW]C2-3-33, salvador¹ chromosome was placed in trans tothe P[EP]3251 (distal to salvador) chromosome in females. Meioticrecombination events were selected for between the two P elements. A SNP57 kb proximal to P[EP]3251 was identified. Some crossovers proximal tothe SNP were salvador⁺, indicating that salvador was proximal to theSNP. Of the salvador⁺ lines, 14 of 19 lines had the polymorphic variantfrom the P[EP]3251 chromosome, while 5 of 19 had the salvador¹chromosome version. Since the SNP was 57 kb away from P[EP]3251,salvador is likely to be located approximately 20 kb proximal to theSNP. Genomic DNA from the salvador chromosomes for five predicted ORFsin this region was sequenced. All these ORFs were found to be wild-typeexcept CG13831, which had a nonsense mutation in each salvadorchromosome.

Microscopy, Immunohistochemistry, Flow Cytometry

For adult eye pictures, sections, and eye SEMs, genotypes were asfollows: y w, eyFLP/+; FRT82B /FRT82B P[mini-w] P[armLacZ] and y w,eyFLP/+; FRT82B salvador^(1/3)/FRT82B P[mini-w] P[armLacZ]. For thoraxSEMs, genotypes were y w, hsFLP/+; FRT82B/FRT82B P[πMyc] P[w y] and y w,hsFLP/+; FRT82B salvador³/FRT82B P[7πMyc] P[w y].

Imaginal disc BrdU incorporations used a 1.5 hr BrdU pulse to visualizeectopic S phases posterior to the MF. Antibodies used wereanti-rabbit-Cy5 and anti-mouse Cy3 (Jackson Laboratories, West Grove,Pa.), a rabbit polyclonal anti-phosH3 antibody (Upstate Laboratories,lake Placid, N.Y.), anti-β-galactosidase rabbit polyclonal (Cappel,Aurora, Ohio), a mouse monoclonal anti-β-galactosidase (Promega,Madison, Wis.), and a mouse monoclonal is anti-DIAP1 antibody and arabbit anti-activated Drice antibody (both provided by Bruce Hay) (Yoo,S. J., et al. (2002) Nat. Cell Biol. 4, 416424). FACS analysis wasperformed as described previously (Neufeld, T. P., et al. (1998) Cell93, 1183-1193; Tapon, N., et al. (2001) Cell 105, 345-355).

For immunofluorescence and TUNEL stainings, discs were dissected fromthe following genotypes: (1) y w, eyFLP/+; FRT82Bsalvador^(1/2/3)/FRT82B P[mini-w] P[armLacZ], (2) y w eyFLP/+; FRT82BWarts^(MGH1)/FRT82B P[mini-w] P[armLacZ], and (3) y w eyFLP/+; FRT82BLATSX1/FRT82B P[mini-w] P[armLacZ]. For TUNEL, DIAP1, or Drice stainingsand adult eye pictures in a GMR-hid transgenic background, genotypeswere y w, eyFLP/+; GMR hid/+; FRT82B/FRT82B P[mini-w] P[armLacZ] and yw, eyFLP/+; GMR-hid/+; FRT82B salvador³/FRT82B P[mini-w] P[armLacZ]. ForDIAP1 or Drice in a GMR-rpr or GMR-hid-Ala5 transgenic background,genotypes were y w, eyFLP/+; GMR-hid/+; FRT82B /FR182B P[mini-w]P]armLacZ] and y w, eyFLP/+; GMR hidAla5 (or 2XGMRrpr)/+; FRT82Bsalvador³/FRT82B P[mini-w] P[UbiGFP]. TUNEL stainings were performed aspreviously described (Kurada, P. and White K. (1998) Cell 95, 319-329).TUNEL positive nuclei were detected with a Rhodamine-conjugated anti-DIGantibody (Boerhinger, Indianapolis, Ind.). For FACS analysis, thegenotype was y w, eyFLP/+; FRT82B salvador³/FRT82B P[mini-w] P[UbiGFP].

Loss-of-function wing clone counts were performed as previouslydescribed (Tapon, N., et al. (2001) Cell 105, 345-355). Clones wereinduced at 48 hr after egg deposition (AED). Discs were dissected foranalysis at 120 hr AED. The genotype was y w, hsFLP/+; FRT82Bsalvador³/FRY82B P[mini-w] P[UbiGFP].

Molecular Biology

The coding region of a salvador cDNA clone was PCR amplified usingoligonucleotide primers with EcoRI and BgIII sites and cloned into pGMR.A 4.1 kb EcoRI/DraI fragment of the warts cDNA (provided by PeterBryant) was cloned into pGMR. GMR-salvador and GMR-warts were thirdchromosome integrations.

Characterization of Human hWW45

The entire coding region of hWW45 was amplified by RT-PCR in twooverlapping fragments. Uncloned PCR products were sequenced directly.The cancer cell lines analyzed is and the DNA used for controlpopulations were performed as described previously (Moberg, K. H., etal. (2001) Nature 413, 311-316).

Primers derived from intronic sequences were used to amplify individualexons of hWW45 from genomic DNA of the ACHN and 780-O cell lines toassess the extent of genomic deletions in these two cases. For theregions flanking hWW45, primers based on nonrepetitive sequences fromBACs containing hWW45 were used.

Protein Binding Studies

The Drosophila warts gene was Myc tagged and cloned into the pCDNA3mammalian expression vector. The Salvador WW domain-GST construct wasgenerated by cloning sequences encoding residues 419495 into theBamHI/EcoRI sites of pGEX-2TK. 293T cells were transfected using Fugeneand harvested 36 hr later in lysis buffer (50 mM Tris-HCl [pH 7.5], 150mM NaCl, 10 mM EDTA, 10% glycerol, 1% Triton X-100, 20 mg/ml leupeptin,10 mg/ml aprotitin, 1 mM PMSF, 0.5 mM DTT, 0.5 mM NaF). Cell lysateswere incubated with 500 ng to 1 mg of GST-fusion protein coupled toglutathione-sepharose, in the presence or absence of peptides (1 mM) for2 hr at 4° C. Peptides were PY-GRQMLPPPPYQSNNN and PAGRQMLPPPPAQSNNN(SEQ ID NO: 25 and SEQ ID NO: 26, respectively). Beads were then washed,treated with protein sample buffer, and subjected to SDS-PAGE. Wartsprotein was detected by immunoblotting with anti-Myc tag 9E10 mAb.

Standard co-immunoprecipitation protocol using 293T cells were used toidentify agents that bind to Salvador molecules, e.g., warts/LATS. Cellswere maintained in Dulbecco's Modified Eagles Medium (DMEM) with 10%Fetal Calf Serum (FCS). Cells were transfected using Fugene (Roche)according to the manufacturer's instructions with the followingplasmids: pCDNA3-HA-Salvador (full-length) and pCDNA3-Myc-warts/LATS(Full-length). Cells were harvested after 48 hours into 400 μl of lysisbuffer (40 mM Tris-HCl pH7.5, 50 mM NaCl, 50 mM NaF, 100 μM NaVO₄, 0.2%NP-40, 10 μg each aprotinin and leupeptin). Insoluble material wasremoved by centrifugation at 4° C. for 10 minutes at 10,000 g. 50 μl oflysate was used per assay. Myc-tagged warts/LATS was precipitated with9E 10 antibodies and 12CA5 was used for HA-tagged Salvador. 20 μl ofprotein A-sepharose were used per assay. The immunoprecipitates werewashed 3 times for 15 minutes with lysis buffer at 4° C., then subjectedto SDS-PAGE (12% gel), transferred to a nitrocellulose filter andanalysed by Western blotting using the same antibodies used for theimmunoprecipitation.

Methods for Screening Human Cells for a Salvador Molecule

To prepare for the Southern Blot Analysis of human tumor cell lines, thefollowing protocol was followed: 10 μg of genomic DNA from samples ofhuman tumor cell lines and control tissues were cut and run on a 0.8-1%agarose gel at 100 V for 4 to 6 hours. Afterwards, a picture was takenwith a ruler next to the gel. The gel was denatured in 1 liter ofdenaturing solution (88 g/L NaCl and 20 g/L NaOH) for 35 to 40 minutesat which point the dye changed color. The gel was rinsed with water andneutralized with 1 liter of neutralizing solution (60.55 g/L Tris baseand 87.6 g/L NaCl, pH 7.2, RT) twice for 20 minutes. The gel wastransferred onto a Hybond N+ membrane (Amarsham Pharmacia, Piscataway,N.J.) with 10×SSC solution (87.65 g/L NaCl, 44.1 g/L sodium citrate, pH7.2, RT). The well positions were marked with a pencil after transfer.The membrane was placed, DNA side up, on a piece of Whatman paper andallowed to absorb the superficial liquid for 20 seconds. The DNA wascrosslinked to a damp membrane using 120,000 μJ using standardautocrosslinking procedures with a Stratagene UV oven. The resultantmembrane, ready for prehybridization, is optionally stored dried betweentwo pieces of Whatman paper for several months in the dark.

The block was prehybridized in 15-20 ml (0.2 ml/cm²) of prehybridizationbuffer at 68° C. for at least 2 hours in a sealed bag. Prehybridizationbuffer contains 6×SSPE and 0.05× blotto. 20×SSPE contains 175.3 g/LNaCl, 27.6 g/L NaH₂PO₄, and 7.4 g/L EDTA, pH 7.4, RT 1× blotto. Blottocontains 5 g of non-fat dried milk powder in 100 ml of water and 0.02%NaN₃, stored at 4° C. 20 ml of prehybridization buffer contains 6 ml ofSSPE 20×, 1 ml of Blotto 1×, and 13 ml of water.

Hybridization proceeded overnight at 68° C. in a sealed bag with 15 mlof hybridizaton solution. Hybridization solution includes 6×SSPE, 0.5%SDS, 100 μg/ml salmon sperm DNA, and no more than 2×10⁶ cpm/ml ofhybridization solution of P³² labeled probe. 15 ml of hybridizationsolution contained 4.5 ml SSPE 20×, 0.375 ml of 20% SDS, 150 ml ofsalmon sperm DNA (10 mg/ml denatured by boiling), 9.975 ml of water, andthe probe, which had been denatured by boiling.

The blot was rinsed for 5 minutes with 50 ml 2×SSPE and 12.5 ml of 20%SCF. The blot was then washed for 15 minutes with 2.5 ml of 20% SDS. Theblot was further washed for 1 hour with 2.5 ml of 20×SSC and 12.5% SDS.The membrane was wrapped in plastic wrap and exposed for at least 16hours at −80° C. with an enhancing screen.

The probe (SEQ ID NO:5) was made on oligolabeling kit (Amersham,Pharmacia). The DNA to be labeled was boiled and 50 ng were added to aneppendorf tube along with 10 μl of reagent mix, 5 μl of a P³² dCTP (3000mCi/mmol), and 1 ml Klenow enzyme. Water was added to bring the volumeto 49 μll. After mixing the reagents, the eppendorf tube was incubatedat 37° C. for 1 hour. After incubation, the unincorporated nucleotideswere discarded by passing the reaction through a Sephadex G50 column.Since 5 μl of a P³² dCTP (3000 mCi/mmol) is equivalent to 100 millioncpm, the signals from the column to that of the reaction were comparedto evaluate the amount of incorporated a P³² dCTP. No more than 2million cpm/ml were placed in the hybridization solution.

All references disclosed herein are incorporated by reference in theirentirety.

1. A method for treating a subject with a disorder characterized byaberrant expression of a salvador molecule, comprising: administering tothe subject an effective amount of a Salvador nucleic acid molecule totreat the disorder.
 2. The method of claim 1, wherein the disorder is acancer.
 3. The method of claim 1, wherein the disorder is an autoimmunedisorder.
 4. The method of claim 1, wherein the disorder is a birthdefect.
 5. A method for treating a subject with a disorder characterizedby aberrant expression of a salvador molecule, comprising: administeringto the subject an effective amount of a Salvador protein to treat thedisorder. 6-8. (canceled)
 9. A method for diagnosing a disordercharacterized by aberrant expression of a Salvador molecule, comprising:detecting in a first biological sample obtained from a subject,expression of a salvador molecule or a Mutant Salvador molecule; whereindecreased expression of the salvador molecule or detectable expressionof the Mutant salvador molecule compared to a control sample indicatesthat the subject has the disorder characterized by aberrant expressionof the salvador molecule. 10-34. (canceled)
 35. An isolated nucleic acidmolecule selected from the group consisting of: (a) nucleic acidmolecules which hybridize under stringent conditions to a nucleic acidmolecule having a nucleotide sequence set forth as SEQ ID NO:3, andwhich code for a Salvador protein, (b) deletions, additions andsubstitutions of the nucleic acid molecules of (a), which code for aSalvador protein, (c) nucleic acid molecules that differ from thenucleic acid molecules of (a) and (b) in codon sequence due to thedegeneracy of the genetic code, and (d) complements of (a), (b) or (c).36. (canceled)
 37. An isolated nucleic acid molecule selected from thegroup consisting of: (a) a unique fragment of the nucleotide sequenceset forth as SEQ ID NO:1 or set forth as SEQ ID NO:3 between 12 and 2000nucleotides in length and (b) complements of (a), wherein the uniquefragments exclude nucleic acids having nucleotide sequences that arecontained within SEQ ID NO:1 or SEQ ID NO:3 and that are known as of thepriority date of this application. 38-40. (canceled)
 41. An isolatednucleic acid molecule selected from the group consisting of: (a) nucleicacid molecules which hybridize under stringent conditions to a nucleicacid molecule having a nucleotide sequence set forth as SEQ ID NO:6, andwhich codes for a Salvador protein, (b) deletions, additions andsubstitutions of the nucleic acid molecules of (a), which code for aSalvador protein, (c) nucleic acid molecules that differ from thenucleic acid molecules of (a) and (b) in codon sequence due to thedegeneracy of the genetic code, and (d) complements of (a), (b) or (c).42. An isolated nucleic acid molecule selected from the group consistingof: (a) a unique fragment of the nucleotide sequence set forth as SEQ IDNO:6 between 12 and 2000 nucleotides in length, and (b) complements of(a), wherein the unique fragments exclude nucleic acids havingnucleotide sequences that are contained within SEQ ID NO:6 and that areknown as of the priority date of this application. 43-44. (canceled) 45.An isolated nucleic acid molecule selected from the group consisting of:(a) nucleic acid molecules which hybridize under stringent conditions toa nucleic acid molecule having a nucleotide sequence set forth as SEQ IDNO:1 or set forth as SEQ ID NO:3, and which code for a Mutant Salvadorprotein, (b) deletions, additions and substitutions of the nucleic acidmolecules of (a), which code for a Mutant Salvador protein, (c) nucleicacid molecules that differ from the nucleic acid molecules of (a) and(b) in codon sequence due to the degeneracy of the genetic code, and (d)complements of (a), (b) or (c).
 46. (canceled)
 47. An expression vectorcomprising the isolated nucleic acid molecule of claim 35 operablylinked to a promoter.
 48. An expression vector comprising isolatednucleic acid molecules selected from the group consisting of: (a)nucleic acid molecules which hybridize under stringent conditions to anucleic acid molecule having a nucleotide sequence set forth as SEQ IDNO:1, and which code for a Salvador protein, (b) deletions, additionsand substitutions of the nucleic acid molecules of (a), which code for aSalvador protein, (c) nucleic acid molecule that differ from the nucleicacid molecules of (a) and (b) in codon sequence due to the degeneracy ofthe genetic code, and (d) complements of (a), (b) or (c), operablylinked to a promoter.
 49. A host cell transformed or transfected withthe expression vector of claim
 48. 50. A transgenic non-human animalcomprising the expression vector of claim
 48. 51-54. (canceled)
 55. Anisolated polypeptide encoded by the isolated nucleic acid molecule ofclaim
 35. 56-85. (canceled)
 86. A method to induce apoptosis in a cell,comprising: administering to the cell an effective amount of a salvadormolecule to induce apoptosis in the cell. 87-90. (canceled)
 91. A methodto inhibit cellular development of a cell comprising: administering tothe cell an effective amount of a salvador molecule to inhibit cellulardevelopment of the cell. 92-95. (canceled)
 96. A method for identifyinga salvador molecule comprising: (a) introducing a putative salvadormolecule or a putative Mutant salvador molecule into a cell; and (b)detecting a salvador functional activity selected from the groupconsisting of binding to a warts/LATS molecule, modulating cellmaturation/differentiation, modulating cell growth, modulating cellproliferation, and modulating cell death. 97-100. (canceled)
 101. Amethod for identifying salvador modulating agents that modulate asalvador molecule-cognate interaction, comprising: (a) contacting asalvador molecule with a cognate under conditions to allow the salvadormolecule to bind to the cognate, in the presence of a putativemodulating agent; wherein the cognate is a warts/LATS molecule; and (b)detecting salvador molecule binding to the cognate; wherein a change insalvador molecule binding to the cognate in the presence of the putativemodulating agent compared to salvador molecule binding to the cognate inthe absence of the cognate indicates that the agent is a salvadormodulating agent.
 102. (canceled)